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Details of the U1 snRNP a, U1 snRNP structure with subunits coloured as in Fig. 1, except for Nam8 (orange), Snu56 (light blue), Snu71 (blue), Luc7 (dark purple), Mud1 (red) and the U1 snRNA (various). The pre-mRNA nucleotides are labelled relative to the first nucleotide (+1) of the intron. The Nam8 RRM1 and RRM2 domains are flexible and project downstream of the 5′SS. The protein attributed to Luc7 in the free U1 snRNP structure¹² was re-assigned to Snu71. C-term, C terminus; N-term, N terminus; SL, stem loop. In the structure we do not observe any evidence that the C-terminal tails of SmB, SmD1, and SmD3 interact with the 5′SS, consistent with their absence in the human 5′SS–minimal U1 snRNP crystal structure¹⁰. b, Representative regions of the sharpened U1 snRNP density determined at 4 Å resolution (map A2) are superimposed on the refined coordinate model. The density reveals side-chain details, and here segments from the Prp42 N terminus (TPR repeat 1), the Sm ring subunit SmB, and the Snu56 α-helical domain are shown. c, The A2 cryo-EM density is shown superimposed on the coordinate models of a selection of U1 snRNP proteins: Luc7, Snu71, Yhc1 and Prp39. In the structure most of Snu71 is disordered, except for a small N-terminal domain (residues 2–43) that binds between the Prp42 N terminus and the Snu56 KH-like fold, consistent with protein crosslinking¹². Functional regions and disordered domains are indicated. d, The U1 snRNA–pre-mRNA 5′ splice site (U1–5′SS) model is superimposed on its cryo-EM density (map A2). A secondary structure diagram of the U1–5′SS interaction is shown underneath the model. The register of the U1–5′SS is shifted by one nucleotide with respect to U1C (Yhc1) compared to the minimal human 5′SS–U1 snRNP crystal structure, owing to an additional nucleotide in the yeast U1 snRNA¹⁰ (U11). Lines indicate Watson–Crick base pairs and dots indicate pseudouridine (ψ)-containing base pairs. e, The Prp39–Prp42 heterodimer is coloured to indicate each of their respective TPR repeats. f, Cryo-EM density of U1 snRNA from maps A2 (dark grey) and A3 (light grey) without (top) and with the superimposed coordinate model of yeast U1 snRNA (bottom). The model is labelled and coloured according to functional regions of U1 snRNA (5′ end, pink; H helix, cyan; SL1, dark blue; SL2-1, green; SL3-1, light blue; SL2-2 and SL3-2 to -7, grey; 3′end and Sm site, yellow). g, Secondary-structure diagram of U1 snRNA. Bold letters indicate residues included in the model, lines indicate Watson–Crick base pairs, and dots G–U wobble and pseudouridine-containing base pairs. Compare to e. The conserved U1 snRNA ‘core’ is outlined with a grey box. The region of the putative phosphate backbone model of part of the U1 SL3-7 region is indicated with a grey box.
Source publication
The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors1. For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2...
Similar publications
Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step...
Citations
... Likewise, RBM39 crosslinks to U2AF, and the LUC7L paralogs LUC7L2 and LUC7L3 (labeled LUC7L) crosslink to several SR proteins. The U1-related protein PRPF40A crosslinks to U1-70K, U1-A and LUC7L3, consistent with previous studies revealing similar interactions of PRP40 in the yeast U1 snRNP in early splicing complexes 45,46 . With the exception of a crosslink between SF3A1 and PRPF40A, crosslinks between U1 and U2 snRNP proteins are not observed, supporting the conclusion that U2 and U1 do not directly contact one another in cross-exon complexes, as previously proposed. ...
Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron¹. Alternatively, it can occur through an exon-defined pathway2–5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5′ splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10–16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5′ splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
... The splicing factor PRPF40A was proposed to mediate a bridging connection already in complex E (prior binding of U2 snRNP) by the simultaneous recognition of U1 snRNP at the 5′ SS and SF1 at the BPS 29,30 . In fact, in budding yeast, this cross-intron connection has been clearly demonstrated by the isolation of the complex E and further characterization by mass spectrometry and cryo-electron microscopy (cryo-EM), showing the involvement of Prp40 (the yeast PRPF40A homolog) in establishing a connection between the 3′and 5′SS by the interaction with Luc7 and Snu71 (from the U1 snRNP) and with Msl5 (yeast SF1) 31,32 . ...
... Recent cryo-EM structures of the early stages of the spliceosome assembly enlighten our knowledge of the molecular details that drive the recognition of the splicing sites that are meant to undergo splicing and the early contacts that the two splicing sites make in these early steps. In yeast, structures of the U1 snRNP 71 , E complex 32 , pre-A complex 19 , and A complex 31 Fig. 6 | Binding selectivity of WW-containing proteins and potential roles of PRPF40A during splicing. A Selectivity mechanisms of WW-containing proteins. ...
PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.
... At the same time, arginine and glutamate residues (REs) and arginine and serine residues (RSs) are located at the C-terminus. This protein is enriched at the 5 ′ end of U1 and U11 snRNAs and binds to the 5 ′ splicing sites (SSs) of pre-mRNAs [20,21]. LUC7L3 has been reported to play a role in human heart failure through the aberrant splicing of sodium channels [22] and to be involved in HBV replication through the regulation of enhancer II/basal core promoter (ENII/BCP) [23]. ...
Alternative splicing has been shown to participate in tumor progression, including hepatocellular carcinoma. The poor prognosis of patients with HCC calls for molecular classification and biomarker identification to facilitate precision medicine. We performed ssGSEA analysis to quantify the pathway activity of RNA splicing in three HCC cohorts. Kaplan–Meier and Cox methods were used for survival analysis. GO and GSEA were performed to analyze pathway enrichment. We confirmed that RNA splicing is significantly correlated with prognosis, and identified an alternative splicing-associated protein LUC7L3 as a potential HCC prognostic biomarker. Further bioinformatics analysis revealed that high LUC7L3 expression indicated a more progressive HCC subtype and worse clinical features. Cell proliferation-related pathways were enriched in HCC patients with high LUC7L3 expression. Consistently, we proved that LUC7L3 knockdown could significantly inhibit cell proliferation and suppress the activation of associated signaling pathways in vitro. In this research, the relevance between RNA splicing and HCC patient prognosis was outlined. Our newly identified biomarker LUC7L3 could provide stratification for patient survival and recurrence risk, facilitating early medical intervention before recurrence or disease progression.
... U2 snRNP belongs to the spliceosome A complex and plays a key role in recognizing the 3ʹ splice site (3ʹ SS), which is essential for pre-mRNA splicing. Furthermore, this recognition process also contributes to the regulation of variable splicing in specific gene pre-mRNA (Plaschka et al. 2018;Wan et al. 2020). ...
Main conclusion
The post-transcriptional gene regulatory pathway and small RNA pathway play important roles in regulating the rapid and long-term response of Rhododendron moulmainense to high-temperature stress.
Abstract
The Rhododendron plays an important role in maintaining ecological balance. However, it is difficult to domesticate for use in urban ecosystems due to their strict optimum growth temperature condition, and its evolution and adaptation are little known. Here, we combined transcriptome and small RNAome to reveal the rapid response and long-term adaptability regulation strategies in Rhododendron moulmainense under high-temperature stress. The post-transcriptional gene regulatory pathway plays important roles in stress response, in which the protein folding pathway is rapidly induced at 4 h after heat stress, and alternative splicing plays an important role in regulating gene expression at 7 days after heat stress. The chloroplasts oxidative damage is the main factor inhibiting photosynthesis efficiency. Through WGCNA analysis, we identified gene association patterns and potential key regulatory genes responsible for maintaining the ROS steady-state under heat stress. Finally, we found that the sRNA synthesis pathway is induced under heat stress. Combined with small RNAome, we found that more miRNAs are significantly changed under long-term heat stress. Furthermore, MYBs might play a central role in target gene interaction network of differentially expressed miRNAs in R. moulmainense under heat stress. MYBs are closely related to ABA, consistently, ABA synthesis and signaling pathways are significantly inhibited, and the change in stomatal aperture is not obvious under heat stress. Taken together, we gained valuable insights into the transplantation and long-term conservation domestication of Rhododendron, and provide genetic resources for genetic modification and molecular breeding to improve heat resistance in Rhododendron.
... In the late steps of nuclear U5 snRNP biogenesis 15,16 , when biogenesis and recycling events become indistinguishable, CD2BP2 and TSSC4 also act on newly made U5 snRNPs through the same mechanisms 9,13,14 . During these biogenesis and recycling steps, the 11-subunit core of the U5 snRNP (U5 snRNA, PRP8, BRR2, SNU114, SNRNP40, seven-subunit Sm ring) 6,11,17,18 incorporates four additional subunits (DIM1, PRP6, DDX23 and USP39) 9,[19][20][21] , and the U4/U6 di-snRNP, which is regenerated independently 22 . Further, essential domains of the U5 snRNP subunits PRP8 and BRR2 arrange into their U4/U6.U5 tri-snRNP conformations [19][20][21] . ...
Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa ‘heart’ of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP–CD2BP2–TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome.
... We analyzed the cryo-EM structure of the yeast spliceosome A complex to identify Luc7 104 amino acids located within 6 Å of the U1 snRNA/5'ss duplex (Fig. 1a, c) (Plaschka et al. 2018). ...
Identification of splice sites is a critical step in pre-mRNA splicing since definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 snRNP. This involves both base pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5’ splice site/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3) which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5’ splice sites. In contrast, the corresponding zinc finger domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5’ splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.
... The 5'-SS binding region of the U11 snRNA is not well resolved in the cryo-EM map, however, a continuous density can be traced from the bottom of the helix H pointing towards the outside of the Sm ring (Fig. 1). This is in agreement with the equivalent region in the U1 snRNP structure (33,36,37). ...
... ; https://doi.org/10.1101/2023.12.22.573053 doi: bioRxiv preprint stabilizes the 5'-SS:U1 snRNP interaction via its two zinc finger domains (37,50,51). In humans, three homologs of Luc7, (LUC7L1, LUC7L2, LUC7L3), are involved in alternative splicing regulation (52,53), differentially affecting distinct sub-classes of the 5'-SS (54). ...
The minor spliceosome catalyses the excision of U12-dependent introns from pre-mRNAs. These introns are rare, but their removal is critical for cell viability. We obtained a cryo-EM reconstruction of the 13-subunit U11 snRNP complex, revealing structures of U11 snRNA and five minor spliceosome-specific factors. U11 snRNP appears strikingly different from the equivalent major spliceosome U1 snRNP. SNRNP25 and SNRNP35 form a dimer, which specifically recognises U11 snRNA. PDCD7 forms extended helices, which bridge SNRNP25 and SNRNP48, located at the distal ends of the particle. SNRNP48 forms multiple interfaces with U11 snRNP and, together with ZMAT5, are positioned near the 5′-end of the U11 snRNA and likely stabilise the binding of the incoming 5′-SS. Our structure provides mechanistic insights into U12-dependent intron recognition and the evolution of the splicing machinery.
... ; https://doi.org/10. 1101 In yeast U1 snRNP, a second protein Luc7 also uses its ZnF2 domain to help stabilizing the interaction between U1 snRNA and the 5' ss (Plaschka et al. 2018;Li et al. 2019). The human homologs (Luc7L, L2, L3) of Luc7 are alternative splicing factors that only transiently associate with U1 snRNP and these proteins contain extra RS rich C-terminal domains that do not exist in yeast Luc7 (Fig. 2A). ...
The recognition of 5-prime splice site (5-prime ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5-prime ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5-prime ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5-prime ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.
... The molecular events leading to stable association of the U2 snRNP with the intron BP are only partially understood. The E-complex, containing pre-mRNA and U1 snRNP Reed 1993, 1991;Li et al. 2019), must merge with a form of the U2 snRNP carrying the SF3a and SF3b proteins (called 17S U2 snRNP in human, Brosi et al. 1993bBrosi et al. , 1993a to recognize the BP and form the prespliceosome or A-complex, within which the 5'ss and BP are base paired to snRNAs U1 and U2, respectively (Zhang et al. 2020b;Plaschka et al. 2018). As the prespliceosome forms, proteins present in E-complex and the U2 snRNP are released to allow new RNA-RNA interactions to be established. ...
Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity.
To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.
... How the spliceosome recognises the correct splice sites remains an important question in biology. In humans, 5′SSs are generally identified first by U1 snRNA through base pairing (1,2), which is followed by the U5 snRNA binding to the 5′SS exon sequences -3 to -1 (3,4), and U6 snRNA binding to the 5′SS intron sequences +3 to +5 (5). The 3′SS recognition is facilitated by the coordinated binding of the U2AF65 -U2AF35 heterodimer to the 3′SS and the SF1/BBP and U2 snRNA to the branch point sequence (6,7). ...
pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5′ ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5′ splice sites. Furthermore, weaker 5′ splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3′ splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5′ splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3′ trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans.
