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Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA represson
Abstract
MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.
Supplementary resource (1)
Crystallographic Data
March 2014
... Indeed, as shown for other DExH/D box proteins (Shibuya et al., 2004;Fairman et al., 2004;Bowers et al., 2006), DDX6 could bind stably to the RNA like a clamp or place holder when bound to ATP. This could prevent RNA-binding proteins from binding mRNAs until the interaction of CNOT1 with DDX6, which promotes ATP hydrolysis and subsequent DDX6 release from RNA (Hondele et al., 2019;Mathys et al., 2014). ...
... To gain insight into how DDX6 limits SGs, we constructed a series of point mutations in DDX6, based on prior work on DDX6, or the highly conserved yeast ortholog DHH1. All generated mutations are conserved between both proteins (Fig. S5 A) and were shown to alter specific protein or RNA interactions or reduce the ATPase activity (Minshall et al., 2009;Dutta et al., 2011;Sharif et al., 2013;Mathys et al., 2014;Ozgur et al., 2015;Kamenska et al., 2016;Brandmann et al., 2018;Balak et al., 2019) (Table 1; and Fig. 6, A and B). We stably expressed these mutants in the DDX6 KO cell line using lentivirus transduction of the DDX6 gene and validated the expression of the DDX6 protein in all constructs via Western blotting or IF (Fig. 6, C and D; and Fig. S5 C). ...
... No PBs with R346A, K352A, K353A mutant in HeLa cells (Ozgur and Stoecklin, 2013) No PBs, increased SGs Mut4 (R373A,T391A R421A) Impairs RNA binding (Dutta et al., 2011), and ATP hydrolysis for DHH1 (Dutta et al., 2011), reduce binding of DDX6 to 4E-T and PAT1B and abolishes binding for LSM14A (Balak et al., 2019) R322A, S340A, R370A in DHH1: Reduction of PBs (Mugler et al., 2016), R322A/S340A in DHH1: Smaller PBs (Dutta et al., 2011), R373Q or T391I missense mutations in DDX6 abolish PBs in patient cells (Balak et al., 2019) No PBs, increased SGs E247A Impairs ATPase activity but is capable of RNA and ATP binding (Dutta et al., 2011) D195A/E196A in DHH1: Increase in size/number of PBs (Dutta et al., 2011), E195Q in DHH1: Constitutive PBs (Mugler et al., 2016) No PBs, increased SGs R386E Impairs CNOT1 binding (Mathys et al., 2014). As CNOT1 can stimulate the RNA-dependent ATPase activity (Mathys et al., 2014), we expect this mutant to behave similarly to the ATPase inactive E247A mutant R55E, F62E, Q282E, N284E, R355E in DHH1: Constitutive PBs (Mugler et al., 2016) No PBs, increased SGs a Like previous observations in DHH1 (Sharif et al., 2013), we noticed a positively charged patch within this mutation containing helix (Fig. S5 B), suggesting partial RNA binding within this area. ...
Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA–RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.
... These constructs were described and characterised previously 16 . We used GFP-trap beads to isolate the recombinant proteins, and analysed protein complexes by Western blotting and bound mRNAs by qPCR. Figure 1C demonstrates efficient isolation of recombinant GFP Ago2, confirms the S387 phosphorylation-dependent increase in Ago2-DDX6 interaction 16 , and further demonstrates that CNOT1, which binds DDX6 directly [29][30][31] , associates with Ago2 in an NMDAR and pS387-dependent manner. In agreement with our hypothesis, the phospho-null mutation (S387A) caused a significant decrease in Limk1 mRNA binding compared to Ago2(WT) under basal conditions, whereas the phospho-mimic (S387D) caused a significant increase (Fig. 1D). ...
... DDX6 associates with RISC via a direct interaction with CNOT1 of the CCR4-NOT complex, which in turn binds GW182, the core scaffold protein that binds directly to Ago2 [29][30][31] . We hypothesized that DDX6 must associate with RISC to mediate the S387 phosphorylation-dependent increase in Limk1 mRNA binding to Ago2. ...
... In addition, since DDX6 is an ATP-dependent helicase, we investigated whether helicase activity is involved in the NMDAR-dependent protein-protein interactions or NMDAR-dependent binding of Limk1 mRNA to RISC, by expressing DDX6 containing a well characterized mutation (E247Q) that blocks enzymatic activity 30,34 . The E247Q mutation had no significant effect on mCherry DDX6-CNOT1 interactions under basal or stimulated conditions, but attenuated the NMDAR-dependent increase in mCherry DDX6 association with Ago2 (Fig. 5A). ...
MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) to modulate protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by repressing the translation of proteins involved in dendritic spine morphogenesis. Rapid NMDAR-dependent silencing of Limk1 is essential for spine shrinkage and requires Ago2 phosphorylation at S387. Not all gene silencing events are modulated by S387 phosphorylation, and the mechanisms that govern the selection of specific mRNAs for silencing downstream of S387 phosphorylation are unknown. Here, we show that NMDAR-dependent S387 phosphorylation causes a rapid and transient increase in the association of Ago2 with Limk1, but not Apt1 mRNA. The specific increase in Limk1 mRNA binding to Ago2 requires recruitment of the helicase DDX6 to RISC. Furthermore, we show that DDX6 is required for NMDAR-dependent silencing of Limk1 via miR-134, but not Apt1 via miR-138, and is essential for NMDAR-dependent spine shrinkage. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression of specific genes to control dendritic spine morphology.
... (C) Available structures for the Ccr4-Not complex. Indicated are the N-terminal module composed of the N-terminal region of CNOT1, CNOT10 (light orange) and CNOT11 (dark orange), PDB entry: 8BFI (Mauxion et al., 2023); MIF4G-like domain 1 of CNOT1, PDB entry: 4J8S (Fabian et al., 2013); the nuclease module composed of the CNOT1 MIF4G domain, Caf1/CNOT7 (light yellow) and Ccr4/CNOT6L (dark yellow), PDB entries 3NGQ and 7VOI (Wang et al., 2010;Zhang et al., 2022); the CNOT9 module, PDB entries 4CT6 or 4CRV (Chen et al., 2014;Mathys et al., 2014) composed of the DUF3819 domain of CNOT1 and CNOT9 (green); a second MIF4G-like domain of CNOT1 modelled using AlphaFold (Jumper et al., 2021); and the NOT module composed of the CNOT1 C-terminal domain and the conserved NOT-Box regions located at the C-termini of CNOT2 (light blue) and CNOT3 (blue), PDB entry: 4C0D . Colours correspond to subunits in panel (B). ...
... CNOT9 is not catalytically active, but structural evidence has shown that it is a hotspot for protein-protein interactions. The interaction of CNOT9 and CNOT1 DUF3819 reveals W-binding pockets on the convex side that can interact with specific tryptophan residues of tristetraprolin (TTP) and GW182/TNRC6 proteins (Chen et al., 2014;Mathys et al., 2014). The armadillo repeats also provide a peptide-binding pocket on the concave side that can accommodate RNA-binding proteins such as Roquin and Bagof-marbles (Sgromo et al., 2017;Sgromo et al., 2018), as well as the conserved CBM of the E3 ubiquitin ligase CNOT4 (Keskeny et al., 2019), thus conferring an important regulatory role. ...
... For example, the MIF4G domain of CNOT1 binds DDX6 (Dhh1/RCK/ p54). This protein is an RNA helicase involved in miRNA regulation, which also activates the decapping pathway and represses translation (Chen et al., 2014;Mathys et al., 2014). Thus, multiple transient, low-affinity interactions between components of the 5′-3′ degradation pathway may result in selforganisation of factors involved in RNA degradation. ...
In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5′cap structure and 3′poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5′-3′ degradation pathway can be induced by trans-acting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5′cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5′-3′ exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved.
... non-adenine bases are encountered in the substrate, given the different degree of inhibition displayed in the presence of distinct nonadenine bases by the two nucleases (Chen et al. 2021) and the loss of the helical structure that a pure poly(A) RNA adopts (Tang et al. 2019). The second function of the M-MIF4G domain in the nuclease module is the recruitment of the DEAD box helicase DDX6/Dhh1 that links the Ccr4-Not complex to mRNA decapping (see below) and translational repression (Chen et al. 2014;Mathys et al. 2014;Ozgur et al. 2015). The M-MIF4G domain of Not1 induces an active conformation in the DDX6 helicase, which is important for efficient miRNA repression (Mathys et al. 2014). ...
... The second function of the M-MIF4G domain in the nuclease module is the recruitment of the DEAD box helicase DDX6/Dhh1 that links the Ccr4-Not complex to mRNA decapping (see below) and translational repression (Chen et al. 2014;Mathys et al. 2014;Ozgur et al. 2015). The M-MIF4G domain of Not1 induces an active conformation in the DDX6 helicase, which is important for efficient miRNA repression (Mathys et al. 2014). A superposition of the available structures reveals that the DDX6 helicase is in close spatial proximity to the nuclease domains ( Figure 2). ...
... Additionally, the nuclease module interacts, through Caf1, with the TOB family of antiproliferative proteins (Horiuchi et al. 2009;Hosoda et al. 2011), thereby linking the deadenylation complex to cell cycle regulation and providing a mechanism through which specific RNAs can be recruited to the deadenylase complex. The Ccr4-Not nuclease module is followed by the Caf40 module that consists of the Not1 CNOT9-binding domain (CN9BD) and the CNOT9/Caf40 (Chen et al. 2014;Mathys et al. 2014). On the one hand, CNOT9 can interact with Trp rich sequences, and thereby bind the TNRC6/GW182 protein, enabling direct recruitment of the Ccr4-Not complex to miRNA targets (Chen et al. 2014;Mathys et al. 2014). ...
The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far.
... The MIF4G domain in the middle of NOT1 provides the docking site for the two catalytic subunits: CAF1 (encoded by Pop2 in Drosophila) is bound directly, whereas CCR4 (twin) binds CAF1 (31) and thus associates with NOT1 indirectly. CAF40 (Rcd1; CNOT9 in humans), which also has affinity for RNA (27,32), associates with the CNOT9 binding domain (CN9BD) of NOT1, which neighbors the MIF4G domain on the Cterminal side (33,34). A NOT2·NOT3 heterodimer (NOT2 encoded by Regena) bound to a C-terminal fragment of NOT1 is termed the NOT module (35,36) (earlier work on the structure of CCR4-NOT reviewed by Wahle and Winkler (7)). ...
... A third constituent of the Smaug-dependent repressor complex is Me31B (DDX6 in mammals) (68,73,81). Me31B associates with the MIF4G domain of NOT1 on a surface opposite the CAF1 binding site (33,34,82), but Me31Bdependent deadenylation has, to our knowledge, not been reported. Me31B also binds Cup, suggesting the possibility that Cup-dependent deadenylation might be mediated by Me31B (83)(84)(85)(86). ...
... The other components of the repressor complex did not affect deadenylation in our in vitro assays. The inactive components included Me31B, even though the protein is able to bind NOT1 (33,34,82). ...
Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug, which binds to Smaug recognition elements (SREs) in the nanos 3'-UTR and nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has a minor inhibitory effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.
... Interestingly, tethering RNF219 strongly repressed the RL reporter mRNA expression compared to the NHA-LacZ control (RL/FL ratio: RNF219 M = 0.307 SD = 0.0624, LacZ M = 5.97 SD = 0.276; two tailed t test: t(2.2) = 34.5, p < 0.001; Fig. 3B). RNF219-mediated repression was higher than repression mediated by the CCR4-NOT subunit CNOT7 and the CNOT1-R fragment 26 (Fig. 3B) previously described 26 Fig. 3B, last bar). This suggests that NHA-RNF219 acts in cis in the tethering assay. ...
... Interestingly, tethering RNF219 strongly repressed the RL reporter mRNA expression compared to the NHA-LacZ control (RL/FL ratio: RNF219 M = 0.307 SD = 0.0624, LacZ M = 5.97 SD = 0.276; two tailed t test: t(2.2) = 34.5, p < 0.001; Fig. 3B). RNF219-mediated repression was higher than repression mediated by the CCR4-NOT subunit CNOT7 and the CNOT1-R fragment 26 (Fig. 3B) previously described 26 Fig. 3B, last bar). This suggests that NHA-RNF219 acts in cis in the tethering assay. ...
... RNF219 affects the poly(A) tail length of a targeted mRNA. The CCR4-NOT complex can repress translation either through its associated deadenylation activity or through the DDX6 pathway acting at the 5′ end of mRNA 26,27 . Several CCR4-NOT associated E3 ligases have been reported to increase deadenylation activity leading to accelerated mRNA decay 51,52 . ...
Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.
... miRNA-target duplexes are bound by Argonaute (Ago) to form the RNA-induced silencing complexes (RISCs) (Pratt and MacRae 2009). Adaptor proteins known such as the GW182 family bind Ago and recruit the PAN2/3 or CNOT complexes to elicit DNA deadenylation and mRNA decay (Braun et al. 2011;Christie et al. 2013;Chen et al. 2014;Mathys et al. 2014). RNA helicase DDX6, an essential downstream effector in the miRNA-mediated translational repression pathway, is recruited to the RISC-GW182-CNOT complex to enhance deadenylase activity Mathys et al. 2014). ...
... Adaptor proteins known such as the GW182 family bind Ago and recruit the PAN2/3 or CNOT complexes to elicit DNA deadenylation and mRNA decay (Braun et al. 2011;Christie et al. 2013;Chen et al. 2014;Mathys et al. 2014). RNA helicase DDX6, an essential downstream effector in the miRNA-mediated translational repression pathway, is recruited to the RISC-GW182-CNOT complex to enhance deadenylase activity Mathys et al. 2014). eIF4E-binding protein 4E-T has also been reported to bind to miRNA-mediated destabilizing transcripts and recruit CNOT deadenylases. ...
RNA deadenylation, the process of shortening of the 3′ poly(A) tail of an RNA molecule, is one of the key steps of post-transcriptional regulation of gene expression in eukaryotic cells. PAN2/3 and CCR4-NOT (CNOT) are the two dominant RNA deadenylation complexes, which play central roles in mediating mRNA decay and translation. While degradation is the final fate of virtually all RNAs in their life cycles, selection of RNA targets as well as control of the rate and timing of RNA decay, in coordination with other molecular pathways, including translation, can be modulated in certain contexts. Such regulation influences cell growth, proliferation, and differentiation at the cellular level; and contributes to establish polarity and regulate signaling at the tissue level. Dysregulation of deadenylation processes have also been implicated in human diseases ranging from cardiac diseases and neurodevelopmental disorders to cancers. In this review, we will discuss mechanisms of gene expression control mediated by the RNA deadenylation complexes and highlight relevant evidence supporting the emerging roles of RNA deadenylation and its regulatory proteins during development and in diseases. A systemic understanding of these mechanisms will be a critical foundation for development of effective strategies to therapeutically target them.
... This result indicates that LegK1 interacts with protein complexes containing Ago1, Ago2 and Ago4 proteins. Interestingly, we also recovered in the Flag-LegK1 immunoprecipitates the TNRC6A, PABPC1 and DDX6 proteins, which are wellcharacterized HMW-miRISC factors [35][36][77][78][79] , suggesting that LegK1 interacts with mature Ago-RISCs engaged in RNA target repression. Importantly, the above interactions were maintained with the catalytic mutant LegK1-KA, while they were significantly reduced with the LegK1-3WF mutant (Figure 2a, b). ...
... These observations suggest that LegK1 might additionally use its W-motifs to physically interact with deadenylase complexes. This hypothesis is consistent with our co-immunoprecipitation data showing that LegK1 interacts with DDX6, a direct partner of CNOT1 35,78 . Based on these data, we propose that LegK1 might trigger silencing suppression by altering, at least in part, the function of HMW-RISCs. ...
RNA silencing is a gene silencing mechanism directed by siRNAs and miRNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 suppresses siRNA- and miRNA-activities in human cells. This ability depends on its kinase activity and on a functional tryptophan-dependent Argonaute (Ago)-binding platform. We further show that the capacity of LegK1 to activate NF-kB signaling contributes to silencing suppression, demonstrating a link between effector-mediated NF-kB signaling activation and silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a key role of this effector in virulence. In infected macrophages, the latter activity relies on the genetic targeting of human Ago4, highlighting a novel function of this host factor in antibacterial resistance.
... DDX6/Dhh1 help to build PBs, and Dhh1 ATPase activity regulates condensate material properties DDX6 and its orthologs are important for many aspects of RNA metabolism, including translation repression, miRNA silencing, and mRNA deadenylation and decay [8,39]. They are major contributors to the formation of PBs [39][40][41][42][43] (Figure 1), with knockdown of DDX6 diminishing PBs in human, Arabidopsis thaliana, and yeast cells [43][44][45][46][47], and nuclear dicing-bodies in Arabidopsis [48], whereas DDX6 overexpression increases PB formation [46,49,50]. ...
... DDX6/Dhh1 help to build PBs, and Dhh1 ATPase activity regulates condensate material properties DDX6 and its orthologs are important for many aspects of RNA metabolism, including translation repression, miRNA silencing, and mRNA deadenylation and decay [8,39]. They are major contributors to the formation of PBs [39][40][41][42][43] (Figure 1), with knockdown of DDX6 diminishing PBs in human, Arabidopsis thaliana, and yeast cells [43][44][45][46][47], and nuclear dicing-bodies in Arabidopsis [48], whereas DDX6 overexpression increases PB formation [46,49,50]. Recombinant human DDX6, yeast Dhh1, and their Arabidopsis orthologs RH6/RH8/RH12 form liquid-like condensates [52]. ...
RNA-dependent DEAD-box ATPases (DDXs) are emerging as major regulators of RNA-containing membrane-less organelles (MLOs). On the one hand, oligomerizing DDXs can promote condensate formation ‘in cis’, often using RNA as a scaffold. On the other hand, DDXs can disrupt RNA–RNA and RNA–protein interactions and thereby ‘in trans’ remodel the multivalent interactions underlying MLO formation. In this review, we discuss the best studied examples of DDXs modulating MLOs in cis and in trans. Further, we illustrate how this contributes to the dynamic assembly and turnover of MLOs which might help cells to modulate RNA sequestration and processing in a temporal and spatial manner.
... DDX6 belongs to the DEAD-box RNA helicase family and participates in a variety of biological processes such as RNA metabolism, translation initiation, and pre-mRNA splicing (Smillie and Sommerville 2002). DDX6 can be recruited to the CCR4-NOT complex to repress mRNA translation (Mathys et al. 2014). Di Stefano et al. (2019) also demonstrated that DDX6 is required for the translational suppression of target mRNAs in P-bodies of stem cells. ...
Background
Liver kinase B1 ( LKB1 ) is frequently mutated in lung adenocarcinoma, and its loss contributes to tumor progression.
Methods
To identify LKB1 downstream genes that promote lung adenocarcinoma aggressiveness, we performed bioinformatical analysis using publicly available datasets.
Results
Rab3B was upregulated in LKB1 -depleted lung adenocarcinoma cells and suppressed by LKB1 overexpression. CREB protein was enriched at the promoter of Rab3B in lung cancer cells. Silencing of CREB abrogated the upregulation of Rab3B upon LKB1 loss. Immunohistochemistry revealed the elevated expression of Rab3B in lung adenocarcinomas relative to adjacent normal tissues. Upregulation of Rab3B was significantly associated with lymph node metastasis, advanced tumor stage, and reduced overall survival in lung adenocarcinoma patients. Knockdown of Rab3B suppressed and overexpression of Rab3B promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells in vitro. In a mouse xenograft model, Rab3B depletion restrained and Rab3B overexpression augmented the growth of lung adenocarcinoma tumors. Mechanistically, Rab3B interacted with DDX6 and enhanced its protein stability. Ectopic expression of DDX6 significantly promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells. DDX6 knockdown phenocopied the effects of Rab3B depletion on lung adenocarcinoma cells. Additionally, DDX6 overexpression partially rescued the aggressive phenotype of Rab3B -depleted lung adenocarcinoma cells.
Conclusion
LKB1 deficiency promotes Rab3B upregulation via a CREB-dependent manner. Rab3B interacts with and stabilizes DDX6 protein to accelerate lung adenocarcinoma progression. The Rab3B-DDX6 axis may be potential therapeutic target for lung adenocarcinoma.
... suggesting that translational repression is directed by miRISC. Recent studies indicate that the Carbon Catabolite Repression-Negative On TATA-less (CCR4-NOT complex), recruited by GW182, deadenylates target mRNAs, resulting in the repression of translation initiation [200][201][202][203][204]. The exact mechanism causing the inhibition of protein production is not clear, but in animals, it has been proposed to occur at initiation, elongation, co-translational protein degradation, and premature termination of translation [159,185,203,204]. ...
The eukaryotic protein synthesis process entails intricate stages governed by diverse mechanisms to tightly regulate translation. Translational regulation during stress is pivotal for maintaining cellular homeostasis, ensuring the accurate expression of essential proteins crucial for survival. This selective translational control mechanism is integral to cellular adaptation and resilience under adverse conditions. This review manuscript explores various mechanisms involved in selective translational regulation, focusing on mRNA-specific and global regulatory processes. Key aspects of translational control include translation initiation, which is often a rate-limiting step, and involves the formation of the eIF4F complex and recruitment of mRNA to ribosomes. Regulation of translation initiation factors, such as eIF4E, eIF4E2, and eIF2, through phosphorylation and interactions with binding proteins, modulates translation efficiency under stress conditions. This review also highlights the control of translation initiation through factors like the eIF4F complex and the ternary complex and also underscores the importance of eIF2{\alpha} phosphorylation in stress granule formation and cellular stress responses. Additionally, the impact of amino acid deprivation, mTOR signaling, and ribosome biogenesis on translation regulation and cellular adaptation to stress is also discussed. Understanding the intricate mechanisms of translational regulation during stress provides insights into cellular adaptation mechanisms and potential therapeutic targets for various diseases, offering valuable avenues for addressing conditions associated with dysregulated protein synthesis.
... In addition, it was reported that Phe1101, Asn1105, Lys1114, Glu1142, Asn1144 and Phe1145 of CNOT1 located at RecA2-binding interface of DDX6. Notably, some structure-based variants on the concave surface of the MIF4G domain specifically weakened the interaction with DDX6 [9,10]. ...
Vissers-Bodmer Syndrome, an autosomal dominant disease, is a neurodevelopmental disorder characterized by global developmental delay, intellectual disability, hypotonia and autistic features with a highly variable phenotype. It is caused by variants in the CCR4-NOT transcription complex, subunit 1 gene (CNOT1). However, the pathophysiologic mechanism of the Vissers-Bodmer Syndrome remains unclear. Notably, this syndrome has not been previously reported in the Chinese. In this study, we utilized whole exome sequencing to identify three novel variants in the CNOT1 gene, encompassing one frameshift variant and two missense variants, in three Chinese patients mainly presenting with developmental delay, intellectual disability and/or autism. Interestingly, three patients exhibited novel manifestations including spina bifida occulta, horse-shoe kidney and café-au-lait spot. The frameshift variant, p.Gly172Alafs*5, occurring de novo, leading to a premature stop codon in the protein, was classified into pathogenic. Two missense variants c.3451A > G (p.Asn1151Asp) and c.557C > T (p.Ser186Phe) were predicted to be deleterious by multiple prediction algorithms with high conservation among a variety of species. Additionally, three-dimensional structure modeling and predicting indicated the substitution of the mutated amino acids would decrease the stability of CNOT1 protein. Given that CNOT1 is a relatively novel disease gene, we evaluated the gene-disease validity following ClinGen Standard Operating Procedure. The existing evidence substantiates a “Definitive” level of gene-disease relationship. The genetic findings provide a reliable basis for the genetic counseling of the family reproduction. Moreover, our results expand the genetic and phenotypic spectrum of CNOT1-related Vissers-Bodmer Syndrome.
... The RecA2 domain of DDX6 mediates multiple protein interactions. It binds the Mid domain of eIF4G (MIF4G) of NOT1 which stimulates its ATPase activity by stabilizing it in an activated conformation (Mathys et al., 2014, Chen et al., 2014. In addition to providing a link to the CCR4-NOT deadenylase complex, the RecA2 domain of DDX6 also mediates interactions with other regulatory proteins e.g. ...
Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrate that the human ortholog of Dhh1p, DDX6, triggers deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation-and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.
... They are abundant in GW182, a glycine-and tryptophan-rich protein of 182 kDa mass (Eystathioy et al. 2002), involved in microRNA-dependent gene silencing. GW182 is responsible for linking the microRNA-targeted mRNA with the CCR4-NOT deadenylase complex by SLIMs' interactions (Fabian et al. 2011;Braun et al. 2013;Mathys et al. 2014). The C-terminal silencing domain (SD) of GW182 was shown experimentally by hydrogen-deuterium exchange mass spectrometry to be intrinsically disordered (Cieplak-Rotowska et al. 2018). ...
Intrinsically disordered proteins (IDPs) form an important class of biomolecules regulating biological processes in higher organisms. The lack of a fixed spatial structure facilitates them to perform their regulatory functions and allows the efficiency of biochemical reactions to be controlled by temperature and the cellular environment. From the biophysical point of view, IDPs are biopolymers with a broad configuration state space and their actual conformation depends on non-covalent interactions of its amino acid side chain groups at given temperature and chemical conditions. Thus, the hydrodynamic radius (Rh) of an IDP of a given polymer length (N) is a sequence- and environment-dependent variable. We have reviewed the literature values of hydrodynamic radii of IDPs determined experimentally by SEC, AUC, PFG NMR, DLS, and FCS, and complement them with our FCS results obtained for a series of protein fragments involved in the regulation of human gene expression. The data collected herein show that the values of hydrodynamic radii of IDPs can span the full space between the folded globular and denatured proteins in the Rh(N) diagram.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00249-023-01683-8.
... These remarkable proteins are largely unstructured and bind with two tryptophans (Ws) into specific binding pockets located on the surface of the PIWI domain of Ago proteins (Elkayam et al. 2017;Pfaff et al. 2013;Schirle and MacRae 2012). TNRC6 proteins form a large scaffold and directly bind to the poly(A) binding protein on the poly(A) of the target mRNA and also recruit deadenylases such as the CCR4-NOT complex or PAN2/3 to the mRNA (Braun et al. 2011;Chekulaeva et al. 2011;Fabian et al. 2009;Huntzinger et al. 2010Huntzinger et al. , 2012Mathys et al. 2014). Subsequent deadenylation leads to recruitment of the decapping complex to the 5′ cap (Rehwinkel et al. 2005). ...
MicroRNA (miRNA)-guided gene silencing is a key regulatory process in various organisms and linked to many human diseases. MiRNAs are processed from precursor molecules and associate with Argonaute proteins to repress the expression of complementary target mRNAs. Excellent work by numerous labs has contributed to a detailed understanding of the mechanisms of miRNA function. However, miRNA effects have mostly been analyzed and viewed as isolated events and their natural environment as part of complex RNA-protein particles (RNPs) is often neglected. RNA binding proteins (RBPs) regulate key enzymes of the miRNA processing machinery and furthermore RBPs or readers of RNA modifications may modulate miRNA activity on mRNAs. Such proteins may function similarly to miRNAs and add their own contributions to the overall expression level of a particular gene. Therefore, post-transcriptional gene regulation might be more the sum of individual regulatory events and should be viewed as part of a dynamic and complex RNP world.
... The role of this amino acid is yet unclear. However, stably associated free tryptophan residues have been described to play allosteric or structural roles in other proteins (42,43). ...
Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo–electron microscopy (cryo-EM) G. mellonella ’s saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins’ functionalities, their role in vivo, and their eventual biotechnological application.
... The role of this amino acid is yet unclear. However, stably associated free tryptophan residues have been described to play allosteric or structural roles in other proteins (42,43). ...
Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo–electron microscopy (cryo-EM) G. mellonella’s saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins’ functionalities, their role in vivo, and their eventual biotechnological application.
... Fourth, the precise mechanisms by which the CCR4-NOT impacts EGFR-and CDK12/13-regulated 4E-BP1 activity and downstream oncoprotein stability remain to be defined. The CCR4-NOT complex has been shown to regulate mRNA metabolism directly through miRNA-mediated deadenylation of mRNAs and translation by interacting with translational regulators such as eIF4E and DDX6 and blocking the decapping machinery (65)(66)(67)(68)(69)(70)(71). Further, it also functions in the ubiquitination of nascent, translationally arrested polypeptides and the maintenance of 26S proteasome integrity (66,72), suggesting that its regulatory roles in the phenomena under study here may be multifactorial, including at transcriptional level. ...
Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.
... In addition, it has been demonstrated that the recruitment of the CCR4-NOT complex to transcripts can lead to targeted translational repression independently of mRNA degradation. In fact, several laboratories have demonstrated that miRNA-induced translation repression of mRNAs occurs via the recruitment of the CCR4-NOT complex (26)(27)(28). Of note, there are likely several additional mechanisms that might contribute to translational repression of NMD targets, for example, through the cap-binding protein 4E-T (29,30). ...
Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1 (UPF1). When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1 : R3H-domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in a SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ-domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.
... On the contrary, DCP1A only partially overlapped with GW182-positi v e granules in control and siPATL1-treated cells and was mainly excluded from GW182-granules in siCNOT1-and si4E-T-treated cells (Supplementary Figure S10). All together, these results indicate that proper PB and SG separation also depends on DDX6 interacting partners CNOT1 and 4E-T that presumably act by modulation of DDX6 helicase / ATPase activity as previously shown ( 81 ). ...
Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.
... In particular, many proteins implicated in mRNA turnover that constitute the cytoplasmic membrane-less organelles 4 protein of 182 kDa mass (Eystathioy et al. 2002), involved in microRNA-dependent gene silencing. GW182 is responsible for linking the microRNA-targeted mRNA with the CCR4-NOT deadenylase complex by SLIMs interactions (Fabian et al. 2011;Braun et al. 2013;Mathys et al. 2014). The C-terminal silencing domain (SD) of GW182 was shown experimentally by hydrogen-deuterium exchange mass spectrometry to be intrinsically disordered (Cieplak-Rotowska et al. 2018). ...
Intrinsically disordered proteins (IDPs) form an important class of biomolecules regulating biological processes in higher organisms. The lack of a fixed spatial structure facilitates them to perform their regulatory functions. Due to the possibility of large conformational changes of IDPs, the cellular milieu can also control productivity of biochemical reactions. From the biophysical point of view, IDPs are biopolymers with a broad configuration state space. The conformation of such a biopolymer depends on non-covalent interactions of its amino acid side chain groups at given temperature and chemical conditions. Thus, the hydrodynamic radius (Rh) of an IDP of a given polymer length (N) is a sequence- and environment-dependent variable. We have reviewed the literature values of hydrodynamic radii of IDPs determined experimentally by SEC, AUC, PFG NMR, DLS, and FCS, and complement them with our FCS results obtained for a series of protein fragments involved in regulation of human gene expression. The data collected herein show that the values of hydrodynamic radii of intrinsically disordered proteins can span the full space between the folded globular and denatured proteins in the Rh(N) diagram.
... 17). For translational repression, GW182 recruits carbon catabolite repressor protein 4 complexes which in turn recruit RNA helicases like DDX6 ( Refs 18,19). MicroRNA-mediated inhibition of mRNA translation initiation results from the interference with the eukaryotic initiation factors eIF4A-I and eIF4A-II (Ref. ...
Cancer metastasis is the primary cause of cancer-related deaths. The seeding of primary tumours at a secondary site is a highly inefficient process requiring substantial alterations in the genetic architecture of cancer cells. These alterations include significant changes in global gene expression patterns. MicroRNAs are small, non-protein coding RNAs which play a central role in regulating gene expression. Here, we focus on microRNA determinants of cancer metastasis and examine microRNA dysregulation in metastatic cancer cells. We dissect the metastatic process in a step-wise manner and summarise the involvement of microRNAs at each step. We also discuss the advantages and limitations of different microRNA-based strategies that have been used to target metastasis in pre-clinical models. Finally, we highlight current clinical trials that use microRNA-based therapies to target advanced or metastatic tumours.
... We previously isolated two DEAD-box RNA helicase genes in Brassica under environmental stress, both are homologs of DDX6 (DEAD-box RNA helicase 6) in animals and RH6 (RNA helicase 6) in Arabidopsis. Recent studies show that DDX6 and AtRH6 are involved in RNA turnover and miRNA biogenesis [17][18][19], but whether they function in plant salt stress response remains unknown. Here, we identified an uncharacterized locus encoding a DDX6 homolog gene BnRH6 in Brassica. ...
Rapeseed (Brassica napus) is one of the most important vegetable oil crops worldwide. Abiotic stresses such as salinity are great challenges for its growth and productivity. DEAD-box RNA helicase 6 (RH6) is a subfamily member of superfamily 2 (SF2), which plays crucial roles in plant growth and development. However, no report is available on RH6 in regulating plant abiotic stress response. This study investigated the function and regulatory mechanism for BnRH6. BnRH6 was targeted to the nucleus and cytoplasmic processing body (P-body), constitutively expressed throughout the lifespan, and induced by salt stress. Transgenic overexpressing BnRH6 in Brassica and Arabidopsis displayed salt hypersensitivity, manifested by lagging seed germination (decreased to 55%–85% of wild-type), growth stunt, leaf chlorosis, oxidative stress, and over-accumulation of Na ions with the K+/Na+ ratio being decreased by 18.3%–28.6%. Given the undesirable quality of knockout Brassica plants, we utilized an Arabidopsis T-DNA insertion mutant rh6-1 to investigate downstream genes by transcriptomics. We constructed four libraries with three biological replicates to investigate global downstream genes by RNA sequencing. Genome-wide analysis of differentially expressed genes (DEGs) (2-fold, p < 0.05) showed that 41 genes were upregulated and 66 genes were downregulated in rh6-1 relative to wild-type under salt stress. Most of them are well-identified and involved in transcription factors, ABA-responsive genes, and detoxified components or antioxidants. Our research suggests that BnRH6 can regulate a group of salt-tolerance genes to negatively promote Brassica adaptation to salt stress.
... Among these proteins, CNOT6, 7, and 8 are subunits of the CCR4-NOT core transcriptional and translational regulation complexes, and are important for the stabilization, cytoplasmic transport, and deadenylation of polyadenylated mRNAs. CNOT6 (CCR4a) and CNOT7 (CAF1) have RNase activity and bind to CNOT1, which is a platform that is able to bind further regulatory proteins [17]. ABCE1 is a highly conserved protein required for translation initiation and ribosome biogenesis, and is a ribonuclease L inhibitor; it also plays a role in translation termination and ribosome recycling by dissociating ribosomes into large and small subunits [18]. ...
The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with Renilla and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the Renilla gene contained sequences targeted by microRNAs (miRNAs) in the 3′UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter Renilla luciferase synthesis. The mRNA-protein complexes formed by transcripts regulated by different miRNAs differed from each other and were different in different cell types, as shown by sucrose gradient centrifugation. Unexpectedly, the presence of miRNA targets on Renilla transcripts also affected the expression of the co-transfected but non-targeted firefly luciferase gene in both cell types. Renilla and firefly transcripts were found in the same sucrose gradient fractions and specific anti-miRNA oligoribonucleotides, which influenced the expression of the Renilla gene, and also influenced that of firefly gene. These results suggest that, in addition to targeted transcripts, miRNAs may also modulate the expression of non-targeted transcripts, and using the latter to normalize the results may cause bias. We discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.
... The CCR4-NOT complex, a predominant generic deadenylase [84], cooperates with miRISC by binding to GW182/TNRC6 protein [80], and then recruits DDX6. The binding of CNOT1 and DDX6 activates the ATPase activity of DDX6, and this change is significant for target gene repression [85]. Our study is connected to previous studies which demonstrated the important role of miRNAs during early embryogenesis. ...
The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 knockout (KO) mouse embryos and employing an in vitro epiblast-like cell (EpiLC) induction system. Our study unveils that DDX6 is an important BMP signaling regulator. Deletion of Ddx6 causes the aberrant upregulation of the negative regulators of BMP signaling, which is accompanied by enhanced expression of Nodal and related genes. Ddx6 KO pluripotent cells acquire higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal and Eomes expression in the primitive streak likely promotes endoderm cell fate specification while inhibiting mesoderm differentiation. We also genetically dissected major DDX6 pathways by generating Dgcr8 , Dcp2 , and Eif4enif1 KO models in addition to Ddx6 KO. We found that the miRNA pathway mutant Dgcr8 KO phenocopies Ddx6 KO, indicating that DDX6 mostly works along with the miRNA pathway during early development, whereas its P-body-related functions are dispensable. Therefore, we conclude that DDX6 prevents aberrant upregulation of BMP signaling inhibitors by participating in miRNA-mediated gene silencing processes. Overall, this study delineates how DDX6 affects the development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.
... Translational repression and deadenylation are two processes interconnected since TNRC6 proteins direct both effects through their interaction with the CCR4-NOT deadenylase complex Chen et al., 2014;Mathys et al., 2014). Several mechanisms of miRNA-mediated translational repression have been proposed Fabian and Sonenberg, 2012;Jonas and Izaurralde, 2015;Iwakawa and Tomari, 2015). ...
RNA interference (RNAi) is an ancestral gene silencing mechanism orchestrated by short non-coding small RNAs, including microRNAs (miRNAs). miRNAs are present in a wide range of eukaryotic organisms and have been characterized in diverse biological processes. During the past decade, plant and mammalian miRNAs have emerged as major regulators of host-bacteria interactions by controlling multiple steps of bacterial infections. As a counter-defense mechanism, type III-secreted effectors from a phytopathogenic Pseudomonas syringae strain were found to suppress different steps of the plant miRNA pathway to enable disease. However, it remains unknown whether mammalian pathogenic bacteria could have evolved similar strategies. Here, we report that the Legionella pneumophila type IV-secreted effector LegK1 efficiently suppresses miRNA activities in human cells. This phenomenon requires both its known eukaryotic-like serine/threonine kinase activity and a newly identified Argonaute (Ago)-binding platform. We found that LegK1 not only interacts with human Ago1, Ago2 and Ago4 but also with other components of the miRNA-induced silencing complex (miRISC). Furthermore, LegK1 was found to promote L. pneumophila growth in both its natural host amoeba and in human macrophages, highlighting its biological relevance in bacterial pathogenesis. Finally, we demonstrated that human Ago4 is a major genetic target of LegK1, whose targeting is required to promote growth of L. pneumophila in human macrophages. Altogether, these findings provide the first evidence that a human pathogenic bacterium can directly suppress RNAi to promote pathogenicity.
... In particular, we observed a concerted upregulation of the RISC effector proteins Ddx6, Dcp1a and Edc4 and especially Ago1 and Ago2 proteins upon Stau2 depletion ( Figure 1B, Supplementary Figure S1A). The depicted protein interactor scheme (Figure 1B inset) suggests that these significantly upregulated RBPs are functionally connected with each other (23,40). This upregulation appears to be predominantly regulated at the protein level, as there was no corresponding significant increase of Ago1 and Ago2 mRNA levels in cortical neurons deficient for Stau2 ( Figure 1C). ...
Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with target mRNAs results in ribonucleoprotein (RNP) assembly involved in translational silencing or RNA degradation. Yet, the dynamics of RNP assembly and its underlying functional implications are unknown. Here, we have characterized the role of the RNA-binding protein Staufen2, a candidate Ago interactor, in RNP assembly. Staufen2 depletion resulted in the upregulation of Ago1/2 and the RISC effector proteins Ddx6 and Dcp1a. This upregulation was accompanied by the displacement of Ago1/2 from processing bodies, large RNPs implicated in RNA storage, and subsequent association of Ago2 with polysomes. In parallel, Staufen2 deficiency decreased global translation and increased dendritic branching. As the observed phenotypes can be rescued by Ago1/2 knockdown, we propose a working model in which both Staufen2 and Ago proteins depend on each other and contribute to neuronal homeostasis.
... Access to the 5'-terminal cap structure is attributed to 4E-T, one of the decapping machineries, which is bound to eIF4E and CNOT1, constituting CCR4-NOT, resulting in circularization of mRNA targeted for decay [12]. The physical link between CNOT1 and DDX6, a decapping activator, was also reported [13]. DDX6 functions to enhance mRNA decay through decapping [14] and constitutes processing bodies (PBs) along with 4E-T [15]. ...
In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.
Me31B, an evolutionarily conserved ATP-dependent RNA helicase, plays an important role in the development of the germline across diverse animal species. Its cellular functionality has been posited as a translational repressor, participating in various RNA metabolism pathways to intricately regulate the spatiotemporal expression of RNAs. Despite its evident significance, the precise role and mechanistic underpinnings of Me31B remain insufficiently understood. This article endeavors to comprehensively review historic and recent research on Me31B, distill the major findings, discern generalizable patterns in Me31B's functions across different research contexts, and provide insights into its fundamental role and mechanism of action. The primary focus of this article centers on elucidating the role of Drosophila Me31B within the germline, while concurrently delving into pertinent research on its orthologs within other species and cellular systems.
Most eukaryotic mRNAs and different non‐coding RNAs undergo a form of 3′ end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)‐specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non‐canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3′ end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context‐dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem‐loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology.
This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution
RNA Processing > 3′ End Processing
RNA Turnover and Surveillance > Regulation of RNA Stability
Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.
The Ccr4‐Not complex is a global regulator of mRNA metabolism in eukaryotic cells that is most well‐known to repress gene expression. Delivery of the complex to mRNAs through a multitude of distinct mechanisms accelerates their decay, yet Ccr4‐Not also plays an important role in co‐translational processes, such as co‐translational association of proteins and delivery of translating mRNAs to organelles. The recent structure of Not5 interacting with the translated ribosome has brought to light that embedded information within the codon sequence can be monitored by recruitment of the Ccr4‐Not complex to elongating ribosomes. Thereby, the Ccr4‐Not complex is empowered with regulatory decisions determining the fate of proteins being synthesized and their encoding mRNAs. This review will focus on the roles of the complex in translation and dynamics of co‐translation events.
This article is categorized under: Translation > Mechanisms
Translation > Regulation
Non-alcoholic fatty liver disease (NAFLD) is associated with human environmental exposure to polychlorinated biphenyls (PCBs). Alternative splicing (AS) is dysregulated in steatotic liver disease and is regulated by splicing factors (SFs) and N-6 methyladenosine (m6A) modification. Here integrated analysis of hepatic mRNA-sequencing data was used to identify differentially expressed SFs and differential AS events (ASEs) in the livers of high fat diet-fed C57BL/6J male mice exposed to Aroclor1260, PCB126, Aroclor1260 + PCB126, or vehicle control. Aroclor1260 + PCB126 co-exposure altered 100 SFs and replicate multivariate analysis of transcript splicing (rMATS) identified 449 ASEs in 366 genes associated with NAFLD pathways. These ASEs were similar to those resulting from experimental perturbations in m6A writers, readers, and erasers. These results demonstrate specific hepatic SF and AS regulatory mechanisms are disrupted by HFD and PCB exposures, contributing to the expression of altered isoforms that may play a role in NAFLD progression to NASH.
RNA helicases are highly conserved proteins that use nucleoside triphosphates to bind or remodel RNA, RNA-protein complexes or both. RNA helicases are classified into the DEAD-box, DEAH/RHA, Ski2-like, Upf1-like and RIG-I families, and are the largest class of enzymes active in eukaryotic RNA metabolism - virtually all aspects of gene expression and its regulation involve RNA helicases. Mutation and dysregulation of these enzymes have been linked to a multitude of diseases, including cancer and neurological disorders. In this Review, we discuss the regulation and functional mechanisms of RNA helicases and their roles in eukaryotic RNA metabolism, including in transcription regulation, pre-mRNA splicing, ribosome assembly, translation and RNA decay. We highlight intriguing models that link helicase structure, mechanisms of function (such as local strand unwinding, translocation, winching, RNA clamping and displacing RNA-binding proteins) and biological roles, including emerging connections between RNA helicases and cellular condensates formed through liquid-liquid phase separation. We also discuss associations of RNA helicases with human diseases and recent efforts towards the design of small-molecule inhibitors of these pivotal regulators of eukaryotic gene expression.
The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.
Accurate and precise regulation of gene expression programmes in eukaryotes involves the coordinated control of transcription, mRNA stability and translation. In recent years, significant progress has been made about the role of sequence elements in the 3′ untranslated region for the regulation of mRNA degradation, and a model has emerged in which recruitment of the Ccr4-Not complex is the critical step in the regulation of mRNA decay. Recruitment of the Ccr4-Not complex to a target mRNA results in deadenylation mediated by the Caf1 and Ccr4 catalytic subunits of the complex. Following deadenylation, the 5′ cap structure is removed, and the mRNA subjected to 5′-3′ degradation. Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is reviewed, with a particular focus on mechanisms of its recruitment to mRNA by sequence motifs in the 3′ untranslated region, codon usage, as well as general mechanisms involving the poly(A) tail.
The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental 10 regulator that functions in mammalian stem cell reprogramming, brain development and 11 cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them 12 through molecular mechanisms that await identification. Here, we uncover that TRIM71 13 silences its targets by recruiting TNRC6/GW182, a core component of the miRNA-14 induced silencing complex (miRISC). Co-immunoprecipitation reveals interaction of 15 TNRC6A with additional RNA-binding proteins and we demonstrate that AGO2, TRIM71, 16 and UPF1 each recruit TNRC6 to specific, largely distinct sets of transcripts to silence 17 them. As cellular TNRC6 levels are limiting, competition occurs among the silencing 18 pathways, such that loss of AGO2 protein, or of AGO2 binding to TNRC6, enhances the 19 activities of the other pathways. We conclude that a miRNA-like silencing activity is 20 shared among different mRNA silencing pathways and that use of TNRC6 as a central 21 hub provides a means to integrate their activities.
The evolutionary conserved CCR4-NOT complex functions in the cytoplasm as the main mRNA deadenylase in both constitutive mRNA turnover and regulated mRNA decay pathways. The versatility of this complex is underpinned by its modular multi-subunit organization, with distinct structural modules actuating different functions. The structure and function of all modules are known, except for that of the N-terminal module. Using different structural approaches, we obtained high-resolution data revealing the architecture of the human N-terminal module composed of CNOT1, CNOT10, and CNOT11. The structure shows how two helical domains of CNOT1 sandwich CNOT10 and CNOT11, leaving the most conserved domain of CNOT11 protruding into solvent as an antenna. We discovered that GGNBP2, a protein identified as a tumor suppressor and spermatogenic factor, is a conserved interacting partner of the CNOT11 antenna domain. Structural and biochemical analyses thus pinpoint the N-terminal CNOT1-CNOT10-CNOT11 module as a conserved protein-protein interaction platform.
RNA-binding proteins (RBPs) regulate essentially every event in the lifetime of an RNA molecule, from its production to its destruction. Whereas much has been learned about RNA sequence specificity and general functions of individual RBPs, the ways in which numerous RBPs instruct a much smaller number of effector molecules, that is, the core engines of RNA processing, as to where, when and how to act remain largely speculative. Here, we survey the known modes of communication between RBPs and their effectors with a particular focus on converging RBP-effector interactions and their roles in reducing the complexity of RNA networks. We discern the emerging unifying principles and discuss their utility in our understanding of RBP function, regulation of biological processes and contribution to human disease.
Different types of small non-coding RNAs, especially microRNAs (miRNAs), may be found in the circulation, either protein bound or enclosed in extracellular vesicles. During gestation, and particular during gestational diabetes mellitus (GDM), the levels of several miRNAs are altered. Worldwide the incidence of GDM is increasing, in part driven by the current obesity epidemic. This is a point of public health concern, because offspring of women with GDM frequently suffer from short and long-term complications of maternal GDM. This has prompted the investigation of whether levels of specific miRNA species, detected early in gestation, may be used as diagnostic or prognostic markers for development of GDM. Here, we summarize mechanisms of RNA secretion, and review circulating miRNAs associated with GDM. Several miRNAs are associated with GDM: miR-29a-3p and miR-29b-3p are generally upregulated in GDM pregnancies, also when measured prior to the development of GDM, while miR-16-5p is consistently upregulated in GDM pregnancies, especially in late gestation. miR-330-3p in circulation is increased in late gestation GDM women, especially in those with poor insulin secretion. miR-17-5p, miR-19a/b-3p, miR-223-3p, miR-155-5p, miR-125-a/b-5p, miR-210-3p and miR-132 are also associated with GDM, but less so and with more contradictory results reported. There could be a publication bias as miRNAs identified early are investigated the most, suggesting that it is likely that additional, more recently detected miRNAs could also be associated with GDM. Thus, circulating miRNAs show potential as biomarkers of GDM diagnosis or prognosis, especially multiple miRNAs containing prediction algorithms show promise, but further studies are needed.
A type of small noncoding RNAs known as microRNAs (miRNAs) fine-tune gene expression posttranscriptionally by binding to certain messenger RNA targets. Numerous physiological processes in the liver, such as differentiation, proliferation, and apoptosis, are regulated by miRNAs. Additionally, there is growing evidence that miRNAs contribute to liver pathology. Extracellular vesicles like exosomes, which contain secreted miRNAs, may facilitate paracrine and endocrine communication between various tissues by changing the gene expression and function of distal cells. The use of stable miRNAs as noninvasive biomarkers was made possible by the discovery of these molecules in body fluids. Circulating miRNAs reflect the conditions of the liver that are abnormal and may serve as new biomarkers for the early detection, prognosis, and evaluation of liver pathological states. miRNAs are appealing therapeutic targets for a range of liver disease states because altered miRNA expression is associated with deregulation of the liver's metabolism, liver damage, liver fibrosis, and tumor formation. This review provides a comprehensive review and update on miRNAs biogenesis pathways and mechanisms of miRNA-mediated gene silencing. It also outlines how miRNAs affect hepatic cell proliferation, death, and regeneration as well as hepatic detoxification. Additionally, it highlights the diverse functions that miRNAs play in the onset and progression of various liver diseases, including nonalcoholic fatty liver disease, alcoholic liver disease, fibrosis, hepatitis C virus infection, and hepatocellular carcinoma. Further, it summarizes the diverse liver-specific miRNAs, illustrating the potential merits and possible caveats of their utilization as noninvasive biomarkers and appealing therapeutic targets for liver illnesses.
The conserved CCR4-NOT complex initiates the decay of mRNAs by catalyzing the shortening of their poly(A) tails in a process known as deadenylation. Recent studies have provided mechanistic insights into the action and regulation of this molecular machine. The two catalytic enzymatic subunits of the complex hydrolyze polyadenosine RNA. Notably, the non-catalytic subunits substantially enhance the complex's affinity and sequence selectivity for polyadenosine by directly contacting the RNA. An additional regulatory mechanism is the active recruitment of the CCR4-NOT to transcripts targeted for decay by RNA-binding proteins that recognize motifs or sequences residing predominantly in untranslated regions. This targeting and strict control of the mRNA deadenylation process emerges as a crucial nexus during post-transcriptional regulation of gene expression.
Decapping is the enzymatic removal of 5′ cap structures from mRNAs in eukaryotic cells. Cap structures normally enhance mRNA translation and stability, and their excision commits an mRNA to complete 5′–3′ exoribonucleolytic digestion and generally ends the physical and functional cellular presence of the mRNA. Decapping plays a pivotal role in eukaryotic cytoplasmic mRNA turnover and is a critical and highly regulated event in multiple 5′–3′ mRNA decay pathways, including general 5′–3′ decay, nonsense‐mediated mRNA decay (NMD), AU‐rich element‐mediated mRNA decay, microRNA‐mediated gene silencing, and targeted transcript‐specific mRNA decay. In the yeast Saccharomyces cerevisiae, mRNA decapping is carried out by a single Dcp1‐Dcp2 decapping enzyme in concert with the accessory activities of specific regulators commonly known as decapping activators or enhancers. These regulatory proteins include the general decapping activators Edc1, 2, and 3, Dhh1, Scd6, Pat1, and the Lsm1‐7 complex, as well as the NMD‐specific factors, Upf1, 2, and 3. Here, we focus on in vivo mRNA decapping regulation in yeast. We summarize recently uncovered molecular mechanisms that control selective targeting of the yeast decapping enzyme and discuss new roles for specific decapping activators in controlling decapping enzyme targeting, assembly of target‐specific decapping complexes, and the monitoring of mRNA translation. Further, we discuss the kinetic contribution of mRNA decapping for overall decay of different substrate mRNAs and highlight experimental evidence pointing to the functional coordination and physical coupling between events in mRNA deadenylation, decapping, and 5′–3′ exoribonucleolytic decay.
GW182 family proteins are a key component of microRNA‐protein complex eliciting translational repression and/or degradation of microRNA‐targets. MicroRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4‐NOT complex which accelerates deadenylation and inhibits translation. The carboxyl‐terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4‐NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino‐terminal Argonaute‐binding domain of human GW182/TNRC6A can associate with the CCR4‐NOT complex. This region resides between the two Argonaute‐binding sites and contains reiterated GW/WG‐motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4‐NOT complex. Furthermore, co‐expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4‐NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4‐NOT complex. This article is protected by copyright. All rights reserved.
Biomolecular condensates require suitable control of material properties for their function. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies consisting of out-of-equilibrium condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which are regulated on several levels. Removal of the low-complexity domains (LCDs) of the protein decreases the fluidity of the condensates. Structured RNA leads to a larger fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU). Promotion of the enzymatic ATPase activity of Dhh1 reduces aging of the condensates and the formation of arrested structures, indicating that biochemical activity and material turnover can maintain fluid-like properties over time.
A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized
eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated
the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of
the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues
in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent
of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis
verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent
on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7
deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated
silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.
Significance
The fate of eukaryotic mRNAs is intimately linked to the complement of proteins that associate with them to form mRNA—protein complexes, the so-called messenger ribonucleoprotein particles (mRNPs). Transitions in the architecture of an mRNP lead to specific functional consequences. DEAD-box proteins are key players in orchestrating these structural rearrangements: They associate with RNA in response to ATP binding and dissociate from it upon ATP hydrolysis. In this paper, we have elucidated the molecular mechanisms by which a DEAD-box protein, which in human cells marks spliced mRNPs for a specialized surveillance pathway, is recognized by the MIF4G domain of a splicing factor. This structure shows how a MIF4G domain can act as a negative regulator of DEAD-box ATPase activity.
The Ccr4-Not complex is involved in several aspects of gene expression, including mRNA decay, translational repression and transcription. We determined the 2.8-Å-resolution crystal structure of a 120-kDa core complex of the Saccharomyces cerevisiae Not module comprising the C-terminal arm of Not1, Not2 and Not5. Not1 is a HEAT-repeat scaffold. Not2 and Not5 have extended regions that wrap around Not1 and around their globular domains, the Not boxes. The Not boxes resemble Sm folds and interact with each other with a noncanonical dimerization surface. Disruption of the interactions within the ternary complex has severe effects on growth in vivo. The ternary complex forms a composite surface that binds poly(U) RNA in vitro, with a site at the Not5 Not box. The results suggest that the Not module forms a versatile platform for macromolecular interactions.
Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a
nontranslatable pool or in their degradation. Removal of the 5′ cap structure is a crucial step that commits deadenylated
mRNAs to 5′-to-3′ degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate
in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure
of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain
of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why
the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based
mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete
with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking–mass spectrometry approach
shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest
a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.
Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3' untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4-NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4-NOT complex. A high-resolution crystal structure of the TTP-CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4-NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.
MicroRNA Mechanism
MicroRNAs are small noncoding RNAs that regulate gene expression by binding complementary target messenger RNAs (mRNAs) and repressing their expression through repression of protein translation and mRNA degradation. Meijer et al. (p. 82 ) show that in a HeLa cell system mRNA degradation is a consequence of translational inhibition via the initiation factor eIF4A2.
The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. Here, we show that the Drosophila melanogaster NOT10 and C2orf29 orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. We further investigated the assembly of the D. melanogaster CCR4-NOT complex, and demonstrate that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, we show that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome.
Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation.
Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact
with poly(A)-binding protein (PABP) and the PAN2–PAN3 and CCR4–NOT deadenylase complexes. These interactions are required
for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing
target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent
repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins
to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are
triggered through the interactions of GW182 proteins with PABP and deadenylases.
The CCR4–NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal
of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to decay. The conserved core of the complex consists
of a catalytic module comprising two deadenylases (CAF1/POP2 and CCR4a/b) and the NOT module, which contains at least NOT1,
NOT2 and NOT3. NOT1 bridges the interaction between the two modules and therefore, acts as a scaffold protein for the assembly
of the complex. Here, we present the crystal structures of the CAF1-binding domain of human NOT1 alone and in complex with
CAF1. The NOT1 domain comprises five helical hairpins that adopt an MIF4G (middle portion of eIF4G) fold. This NOT1 MIF4G
domain binds CAF1 through a pre-formed interface and leaves the CAF1 catalytic site fully accessible to RNA substrates. The
conservation of critical structural and interface residues suggests that the NOT1 MIF4G domain adopts a similar fold and interacts
with CAF1 in a similar manner in all eukaryotes. Our findings shed light on the assembly of the CCR4–NOT complex and provide
the basis for dissecting the role of the NOT module in mRNA deadenylation.
Shortening eukaryotic poly(A) tails represses mRNA translation and induces mRNA turnover. The major cytoplasmic deadenylase, the Ccr4-Not complex, is a conserved multisubunit assembly. Ccr4-Not is organized around Not1, a large scaffold protein that recruits two 3'-5' exoribonucleases, Caf1 and Ccr4. We report structural studies showing that the N-terminal arm of yeast Not1 has a HEAT-repeat structure with domains related to the MIF4G fold. A MIF4G domain positioned centrally within the Not1 protein recognizes Caf1, which in turn binds the LRR domain of Ccr4 and tethers the Ccr4 nuclease domain. The interactions that form the nuclease core of the Ccr4-Not complex are evolutionarily conserved. Their specific disruption affects cell growth and mRNA deadenylation and decay in vivo in yeast. Thus, the N-terminal arm of Not1 forms an extended platform reminiscent of scaffolding proteins like eIF4G and CBP80, and places the two nucleases in a pivotal position within the Ccr4-Not complex.
PUF proteins are a conserved family of eukaryotic RNA-binding proteins that regulate specific mRNAs: they control many processes including stem cell proliferation, fertility, and memory formation. PUFs repress protein expression from their target mRNAs but the mechanism by which they do so remains unclear, especially for humans. Humans possess two PUF proteins, PUM1 and PUM2, which exhibit similar RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed functional assays to measure sequence-specific repression by PUM1 and PUM2. Both robustly inhibit translation and promote mRNA degradation. Purified PUM complexes were found to contain subunits of the CCR4-NOT (CNOT) complex, which contains multiple enzymes that catalyze mRNA deadenylation. PUMs interact with the CNOT deadenylase subunits in vitro. We used three approaches to determine the importance of deadenylases for PUM repression. First, dominant-negative mutants of CNOT7 and CNOT8 reduced PUM repression. Second, RNA interference depletion of the deadenylases alleviated PUM repression. Third, the poly(A) tail was necessary for maximal PUM repression. These findings demonstrate a conserved mechanism of PUF-mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase leading to translational inhibition and mRNA degradation. A second, deadenylation independent mechanism was revealed by the finding that PUMs repress an mRNA that lacks a poly(A) tail. Thus, human PUMs are repressors capable of deadenylation-dependent and -independent modes of repression.
Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.
Author Summary
Translation of mRNA into protein and turnover of mRNA are two points at which cells can exert regulatory control of gene expression, thereby ensuring that the protein products are present in cells and tissues at the appropriate time and place. The DDX6 family of DEAD box helicases, exemplified by the yeast protein Dhh1, is a group of well-conserved eukaryotic proteins that regulate translation and mRNA decay. As DDX6 proteins are known to be important for diverse processes such as cellular stress responses, early embryonic development, and replication of some viruses, understanding their mechanism of action could be of broad significance to many fields. Previous studies suggest that Dhh1 and other DDX6-family proteins mainly regulate translation at the initiation stage, triggering sequestration and/or decapping of the mRNA. Our work expands the potential functions of Dhh1, showing that Dhh1 is also capable of inhibiting translation at later stages when ribosomes are already loaded onto mRNAs. This extended function for Dhh1 allows a more robust translational control, as inhibition at a late stage of translation can provide immediate stoppage of protein production, as well as affording the potential for storing mRNA already primed and loaded with ribosomes for subsequent rapid re-utilization.
MicroRNAs (miRNAs) regulate most cellular functions, acting by posttranscriptionally repressing numerous eukaryotic mRNAs. They lead to translational repression, deadenylation and degradation of their target mRNAs. Yet, the relative contributions of these effects are controversial and little is known about the sequence of events occurring during the miRNA-induced response. Using stable human cell lines expressing inducible reporters, we found that translational repression is the dominant effect of miRNAs on newly synthesized targets. This step is followed by mRNA deadenylation and decay, which is the dominant effect at steady state. Our findings have important implications for understanding the mechanism of silencing and reconcile seemingly contradictory data.
Human Argonaute Revealed
RNA interference (RNAi) is mediated by Argonaute (Ago) proteins, which bind small regulatory RNAs that have sequence complementarity to target RNAs destined to be silenced. The structure of bacterial homologs of the Ago proteins, and fragments of eukaryotic Ago proteins, have provided initial insights into Ago function. Now, Schirle and MacRae (p. 1037 , published online 26 April; see the Perspective by Kaya and Doudna ) have determined the structure of the full-length human Ago protein bound to a single-stranded (ss) guide RNA. Within the bilobed structure, eight nucleotides of the ssRNA are visibly positioned in the RNA-guide, strand-binding site. The “seed” region of the ssRNA has its Watson-and-Crick base edges exposed to solvent, likely aiding target recognition. The location of two free tryptophans in the Piwi domain suggests a possible recruitment site for Ago-interacting proteins.
microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger RNA (mRNA) deadenylation and
decay. Because translation, deadenylation, and decay are closely linked processes, it is important to establish their ordering
and thus to define the molecular mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated
gene silencing by using a Drosophila S2 cell-based controllable expression system and show that mRNAs with both natural and engineered 3′ untranslated regions
with miRNA target sites are first subject to translational inhibition, followed by effects on deadenylation and decay. We
next used a natural translational elongation stall to show that miRNA-mediated silencing inhibits translation at an early
step, potentially translation initiation.
AU-rich elements (AREs), residing in the 3′ untranslated region (UTR) of many labile mRNAs, are important cis-acting elements that modulate the stability of these mRNAs by collaborating with trans-acting factors such as tristetraprolin (TTP). AREs also regulate translation, but the underlying mechanism is not fully understood.
Here we examined the function and mechanism of TTP in ARE-mRNA translation. Through a luciferase-based reporter system, we
used knockdown, overexpression, and tethering assays in 293T cells to demonstrate that TTP represses ARE reporter mRNA translation.
Polyribosome fractionation experiments showed that TTP shifts target mRNAs to lighter fractions. In murine RAW264.7 macrophages,
knocking down TTP produces significantly more tumor necrosis factor alpha (TNF-α) than the control, while the corresponding
mRNA level has a marginal change. Furthermore, knockdown of TTP increases the rate of biosynthesis of TNF-α, suggesting that
TTP can exert effects at translational levels. Finally, we demonstrate that the general translational repressor RCK may cooperate
with TTP to regulate ARE-mRNA translation. Collectively, our studies reveal a novel function of TTP in repressing ARE-mRNA
translation and that RCK is a functional partner of TTP in promoting TTP-mediated translational repression.
miRNAs recruit the miRNA-induced silencing complex (miRISC), which includes Argonaute and GW182 as core proteins. GW182 proteins effect translational repression and deadenylation of target mRNAs. However, the molecular mechanisms of GW182-mediated repression remain obscure. We show here that human GW182 independently interacts with the PAN2-PAN3 and CCR4-NOT deadenylase complexes. Interaction of GW182 with CCR4-NOT is mediated by two newly discovered phylogenetically conserved motifs. Although either motif is sufficient to bind CCR4-NOT, only one of them can promote processive deadenylation of target mRNAs. Thus, GW182 serves as both a platform that recruits deadenylases and as a deadenylase coactivator that facilitates the removal of the poly(A) tail by CCR4-NOT.
miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation.
Translation, storage, and degradation of messenger ribonucleic acids (mRNAs) are key steps in the posttranscriptional control of gene expression, but how mRNAs transit between these processes remains poorly understood. In this paper, we functionally characterized the DExD/H box adenosine triphosphatase (ATPase) Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, we showed that Dhh1 was sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression was followed by mRNA decay; however, deleting components of the 5'-3' decay pathway uncoupled these processes. Whereas Dhh1's ATPase activity was not required to induce translational inhibition and mRNA decay when directly tethered to an mRNA, ATP hydrolysis regulated processing body dynamics and the release of Dhh1 from these RNA-protein granules. Our results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored, or decayed.
Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least
some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially
as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function
in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues
have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker
RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions
between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these
interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover,
RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited
into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors
to impart stringent control over this very abundant RNA helicase.
Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.
The CCR4-CAF1-NOT complex is a major cytoplasmic deadenylation complex in yeast and mammals. This complex associates with RNA-binding proteins and microRNAs to repress translation of target mRNAs. We sought to determine how CCR4 and CAF1 participate in repression and control of maternal mRNAs using Xenopus laevis oocytes. We show that Xenopus CCR4 and CAF1 enzymes are active deadenylases and repress translation of an adenylated mRNA. CAF1 also represses translation independent of deadenylation. The deadenylation-independent repression requires a 5' cap structure on the mRNA; however, deadenylation does not. We suggest that mere recruitment of CAF1 is sufficient for repression, independent of deadenylation.
MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3'-untranslated regions to repress protein synthesis by mechanisms that are not fully understood. In this review, we describe principles of miRNA-mRNA interactions and proteins that interact with miRNAs and function in miRNA-mediated repression. We discuss the multiple, often contradictory, mechanisms that miRNAs have been reported to use, which cause translational repression and mRNA decay. We also address the issue of cellular localization of miRNA-mediated events and a role for RNA-binding proteins in activation or relief of miRNA repression.
The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited
to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis.
Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in
RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function
to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding,
using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction
with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role
for the GW/WG repeats as effector motifs in miRNA-mediated repression.
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.
The RNA helicase p54 (DDX6, Dhh1, Me31B, Cgh-1, RCK) is a prototypic component of P-(rocessing) bodies in cells ranging from yeast to human. Previously, we have shown that it is also a component of the large cytoplasmic polyadenylation element-binding protein translation repressor complex in Xenopus oocytes and that when tethered to the 3' untranslated region, Xp54 represses reporter mRNA translation. Here, we examine the role of the p54 helicase activity in translational repression and in P-body formation. Mutagenesis of conserved p54 helicase motifs activates translation in the tethered function assay, reduces accumulation of p54 in P-bodies in HeLa cells, and inhibits its capacity to assemble P-bodies in p54-depleted cells. Similar results were obtained in four helicase motifs implicated in ATP binding and in coupling ATPase and RNA binding activities. This is accompanied by changes in the interaction of the mutant p54 with the oocyte repressor complex components. Surprisingly, the C-terminal D2 domain alone is sufficient for translational repression and complete accumulation in P-bodies, although it is deficient for P-body assembly. We propose a novel RNA helicase model, in which the D2 domain acts as a protein binding platform and the ATPase/helicase activity allows protein complex remodeling that dictates the balance between repressors and an activator of translation.
The Ccr4-Not complex is a global regulator of transcription that affects genes positively and negatively and is thought to regulate transcription factor IID function. Two components of this complex, Caf1p and Ccr4p, are directly involved in mRNA deadenylation, and Caf1p is associated with Dhh1p, a putative RNA helicase thought to be a component of the decapping complex. In this work, we tried to determine whether Dhh1p might interact with the Ccr4-Not complex. We found that, first, not mutations displayed severe synthetic phenotypes when combined with a dhh1-null mutation. Second, overexpression of Not1p was toxic in dhh1-null cells. Third, a not mutant phenotype was suppressed by deletion of DHH1 and mimicked by overexpression of DHH1. Fourth, dhh1-null mutants displayed resistance to heat shock, a phenotype observed for all mutants that affect the Ccr4-Not complex. Finally, like Caf1p and Ccr4p, Dhh1p co-immunoprecipitated with the nonessential N-terminal domain of Not1p, and the levels of Caf1p and Dhh1p were dependent upon this Not1p domain. Taken together, our results suggest that the Ccr4-Not complex, via the N-terminal region of Not1p, is necessary for the maintenance of stable cellular levels of Dhh1p and Caf1p, thus contributing to regulation of mRNA decay in addition to transcription.
The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to eIF4A and mjDEAD, in which no motif interactions exist, in Dhh1p, motif V interacts with motif I and the Q-motif, thereby linking the two domains together. Electrostatic potential mapping combined with mutagenesis reveals that motifs I, V, and VI are involved in RNA binding. In addition, trypsin digestion of tDhh1p suggests that ATP binding enhances an RNA-induced conformational change. Interestingly, some mutations located in the conserved motifs and at the interface between the two Dhh1 domains confer dominant negative phenotypes in vivo and disrupt the conformational switch in vitro. This suggests that this conformational change is required in Dhh1 function and identifies key residues involved in that transition.
RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and
let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.
The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational
fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2α, bind to nascent transcripts on
chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression,
mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one
subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like
Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs
and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have
revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation,
storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages
of their lifetimes marks them out as key regulators of post-transcriptional gene expression.
CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.
The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.
The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor protein crystal structure that, unexpectedly, forms intertwined and asymmetric homodimers. Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a C-terminal knob domain. The PAN3 pseudokinase binds ATP, and this function is required for mRNA degradation in vivo. We further identified conserved surfaces required for mRNA degradation, including the binding surface for the PAN2 deadenylase on the knob domain. The most remarkable structural feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Together, our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.
Latin America, the vast geopolitical region from Mexico to Cape Horn at the tip of South America, evokes images of the rain forests of the Amazon and snow-capped peaks of the Andes, ancient Incan and Mayan civilizations, Spanish and Portuguese colonialism, and iconic cities like Rio de Janeiro. Biological research in Latin America has traditionally focused on disciplines such as physiology, pharmacology, biochemistry, genetics, immunology, and parasitology as well as on agricultural and ecological sciences. However, an energetic group of developmental biologists has recently emerged from Argentina, Brazil, Chile, Ecuador, Mexico, Peru, Uruguay, and Venezuela. In 2003, this group, composed of senior scientists and young Latin American developmental biologists who recently returned to their home countries after training in America and Europe, founded the Latin American Society of Developmental Biology (LASDB). Led by their current president Roberto Mayor (University of Chile, Santiago/University College London), the LASDB (http://www.lasdb.org) is promoting developmental biology research in Latin America.
MicroRNAs (miRNAs) guide RNA-induced silencing complex (RISC) that contains an Argonaute family protein to complementary target messenger RNAs (mRNAs). Via RISC, miRNAs silence the expression of target mRNAs by shortening the poly(A) tail-which leads to mRNA decay-and by repressing translation. It has been suggested that GW182, an Argonaute-associating protein, plays the central role in such microRNA actions. Here we show that, although GW182 is obligatory for poly(A) shortening, translational repression by microRNAs occurs even in the absence of GW182. Yet, GW182 is also capable of inducing translational repression independently. Both of these translational repression mechanisms block formation of 48S and 80S ribosomal complexes. Thus microRNAs utilize at least three distinct silencing pathways: GW182-mediated deadenylation and GW182-dependent and -independent repression of early translation initiation. Differential contribution from these multiple pathways may explain previous, apparently contradictory observations of how microRNAs inhibit protein synthesis.
Translation Block
MicroRNAs (miRNAs) are small, noncoding RNA genes that are found in the genomes of most eukaryotes, where they play an important role in the regulation of gene expression. Although whether gene activity is repressed by blocking translation of messenger RNA (mRNA) targets, or by promoting their deadenylation and then degradation, has been open to debate. Bazzini et al. (p. 233 , published online 15 March) and Djuranovic et al. (p. 237 ) looked at early points in the repression reaction in the zebrafish embryo or in Drosophila tissue culture cells, respectively, and found that translation was blocked before target mRNAs were significantly deadenylated and degraded. Thus, miRNAs appear to interfere with the initiation step of translation.
RNA helicase enzymes catalyze the in vivo folding and conformational re-arrangement of RNA. DEAD-box proteins (DBPs) make up the largest family of RNA helicases and are found across all phyla. DBPs are molecular motor proteins that utilize chemical energy in cycles of ATP binding, hydrolysis, and product release to perform mechanical work resulting in reorganization of cellular RNAs. DBPs contain a highly conserved motor domain helicase core. Auxiliary domains, enzymatic adaptations, and regulatory partner proteins contribute to the diversity of DBP function throughout RNA metabolism. In this review we focus on the current understanding of the DBP ATP utilization mechanism in rearranging and unwinding RNA structures. We discuss DBP structural properties, kinetic pathways, and thermodynamic features of nucleotide-dependent interactions with RNA. We highlight recent advances in the DBP field derived from biochemical and molecular biophysical investigations aimed at developing a quantitative mechanistic understanding of DBP molecular motor function.
miRNAs are posttranscriptional regulators of gene expression that associate with Argonaute and GW182 proteins to repress translation and/or promote mRNA degradation. miRNA-mediated mRNA degradation is initiated by deadenylation, although it is not known whether deadenylases are recruited to the mRNA target directly or by default, as a consequence of a translational block. To answer this question, we performed a screen for potential interactions between the Argonaute and GW182 proteins and subunits of the two cytoplasmic deadenylase complexes. We found that human GW182 proteins recruit the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes through direct interactions with PAN3 and NOT1, respectively. These interactions are critical for silencing and are conserved in D. melanogaster. Our findings reveal that GW182 proteins provide a docking platform through which deadenylase complexes gain access to the poly(A) tail of miRNA targets to promote their deadenylation, and they further indicate that deadenylation is a direct effect of miRNA regulation.
The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5' end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an "open" conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a "closed" conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the "P" site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded "landing pad" for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.
MicroRNAs (miRNAs) regulate gene expression through translation repression and mRNA destabilization. However, the molecular mechanisms of miRNA silencing are still not well defined. Using a genetic screen in mouse embryonic stem (ES) cells, we identify mammalian hyperplastic discs protein EDD, a known E3 ubiquitin ligase, as a key component of the miRNA silencing pathway. ES cells deficient for EDD are defective in miRNA function and exhibit growth defects. We demonstrate that E3 ubiquitin ligase activity is dispensable for EDD function in miRNA silencing. Instead, EDD interacts with GW182 family proteins in the Argonaute-miRNA complexes. The PABC domain of EDD is essential for its silencing function. Through the PABC domain, EDD participates in miRNA silencing by recruiting downstream effectors. Among the PABC-interactors, DDX6 and Tob1/2 are both required and sufficient for silencing mRNA targets. Taken together, these data demonstrate a critical function for EDD in miRNA silencing.
Despite their widespread roles as regulators of gene expression, important questions remain about target regulation by microRNAs. Animal microRNAs were originally thought to repress target translation, with little or no influence on mRNA abundance, whereas the reverse was thought to be true in plants. Now, however, it is clear that microRNAs can induce mRNA degradation in animals and, conversely, translational repression in plants. Recent studies have made important advances in elucidating the relative contributions of these two different modes of target regulation by microRNAs. They have also shed light on the specific mechanisms of target silencing, which, although it differs fundamentally between plants and animals, shares some common features between the two kingdoms.
MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of "off-target" mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.
MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.
The DEAD box helicase DDX6/Me31B functions in translational repression and mRNA decapping. How particular RNA helicases are recruited specifically to distinct functional complexes is poorly understood. We present the crystal structure of the DDX6 C-terminal RecA-like domain bound to a highly conserved FDF sequence motif in the decapping activator EDC3. The FDF peptide adopts an alpha-helical conformation upon binding to DDX6, occupying a shallow groove opposite to the DDX6 surface involved in RNA binding and ATP hydrolysis. Mutagenesis of Me31B shows the relevance of the FDF interaction surface both for Me31B's accumulation in P bodies and for its ability to repress the expression of bound mRNAs. The translational repressor Tral contains a similar FDF motif. Together with mutational and competition studies, the structure reveals why the interactions of Me31B with EDC3 and Tral are mutually exclusive and how the respective decapping and translational repressor complexes might hook onto an mRNA substrate.
miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. In animal cells, degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1, or NOT1. We show that approximately 60% of AGO1 targets are regulated by CAF1 and/or NOT1, indicating that deadenylation is a widespread effect of miRNA regulation. However, neither a poly(A) tail nor mRNA circularization are required for silencing, because mRNAs whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo. We show further that miRNAs trigger mRNA degradation, even when binding by 40S ribosomal subunits is inhibited in cis. These results indicate that miRNAs promote mRNA decay by altering mRNP composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.
Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element‐binding protein (CPEB) co‐immunoprecipitates
with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2–Xp54 represses the translation of non‐adenylated firefly luciferase mRNAs and that mutations
in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild‐type MS2–Xp54 tethered
to the reporter mRNA 3′‐untranslated region (UTR) represses translation in both oocytes and eggs in an RNA‐dependent complex
with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization
is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic
maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent.
Supporting evidence for RNA‐mediated oligomerization of endogenous Xp54, and RNA‐independent association with CPEB and eIF4E
in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the
Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3′UTR, represses mRNA translation, possibly by sequestering eIF4E from
the translational machinery.
Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit translation and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing non-translating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation. Strains lacking both Dhh1p and Pat1p show strong defects in mRNA decapping and P body formation and are blocked in translational repression. Contrastingly, overexpression of Dhh1p or Pat1p causes translational repression, P body formation, and arrests cell growth. Dhh1p, and its human homolog, RCK/p54, repress translation in vitro, and Dhh1p function is bypassed in vivo by inhibition of translational initiation. These results identify a broadly acting mechanism of translational repression that targets mRNAs for decapping and functions in translational control. We propose this mechanism is competitively balanced with translation, and shifting this balance is an important basis of translational control.