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List of proteins bound to U30 RNA in HEK293 cells. (A) A schematic overview of the protocol. FASP stands for filter-aided sample preparation. (B) Western blot analysis of the cell fractionation. Tubulin was used as a cytoplasmic marker, and histone H1, lamin A and ZCCHC9 represented nuclear markers. (C) Protein profiles in control and U30 RNA-bound nuclear and cytoplasmic extracts. Equal amounts of eluates from control RNA (Ctrl) and U30 RNA bait fractions were separated on 12% SDS-PAGE gel and silver stained. (D) List of selected factors accumulated exclusively on the U30 RNA in human and mouse cells (24) in at least one replicate. The factors are grouped based on protein complexes they form. The last two columns display the presence (+) or absence (−) of particular protein in nuclear (NE) or cytoplasmic (CE) fraction, respectively. For full list of proteins see Supplementary Tables S1 and S2. NPC—nuclear pore complex, FACT—facilitates chromatin transcription/transactions complex, Lsm—SM-like genes forming U6 snRNP complex, THO—ribonucleoprotein nuclear export complex, NEXT—nuclear exosome targeting complex.
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The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for...
Citations
... As a functional read-out, we monitored levels of a panel of NEXT RNA substrates in RBM7 WT, S136A/D, and R143A complemented cells. As controls we included the RBM7 F15A/F53A variant, with diagnostic point mutations in the RNA-recognition motif (RRM) domain (43), and also measured RNA levels of OCT4 mRNA, which is not a NEXT substrate. ...
The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α-helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting complex by binding to an α-helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.
One sentence summary
α-helices in PP2A-B55 substrates bind a conserved pocket on B55 through a common mechanism that is conserved in eukaryotes.
... The compacted structures might protect the naked pre-snRNAs and prevent their degradation. Indeed, removal of the Sm site or shortening/misprocessing of the 3′ end leads to uridylation and rapid degradation of snRNAs [86][87][88][89][90] . Compacted pre-snRNA structures might also serve as a checkpoint allowing the SMN complex to discriminate a correct pre-snRNA substrate from a random RNA molecule containing a stretch of uridines. ...
Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3′-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.
... UV crosslinking captures direct RNA-RBP interactions with a slight preference for U-rich RNA sequences 64 . Since the highest density of RBM7 footprints was detected at intron 3′ends, which are inherently U-rich, longer RBM7 dwell times might not be the only reason for such strong cross-linking 65 . However, since sequences immediately downstream of mature snoRNAs are likely to be less Urich, their high-density RBM7 binding likely reflects longer RNA dwell times of the protein, which could represent sequential RBM7-mediated loading of the RNA exosome onto the pre-snoRNA target. ...
Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5′end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3′end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.
... In the human nucleoplasm, the degradation of molecules with an unprotected 3' end, such as promoter upstream transcripts/upstream antisense RNAs (PROMPTs/uaRNAs), bidirectional enhancer RNAs (eRNAs), hTRs, spliced introns and intron-encoded snoR-NAs, is carried out soon after transcription by NEXT, which contains a zinc-finger protein, ZCCHC8, as well as RNA-binding motif protein 7 (RBM7), which has an affinity for polypyrimidines (46,(59)(60)(61)(62)(63). On the other hand, nucleoplasmic degradation of molecules whose 3' ends have been processed and polyadenylated by canonical PAPs is carried out by the PAXT complex, which contains ZFC3H1, a zinc-finger protein, and PABPN1, which has an affinity for polyadenines (47,(64)(65)(66)(67)(68). ...
Recent in vitro reconstitution analyses have proven that the physical interaction between the exosome core and MTR4 helicase, which promotes the exosome activity, is maintained by either MPP6 or RRP6. However, knowledge regarding the function of MPP6 with respect to in vivo exosome activity remains scarce. Here, we demonstrate a facilitative function of MPP6 that composes a specific part of MTR4-dependent substrate decay by the human exosome. Using RNA polymerase II-transcribed poly(A)+ substrate accumulation as an indicator of a perturbed exosome, we found functional redundancy between RRP6 and MPP6 in the decay of these poly(A)+ transcripts. MTR4 binding to the exosome core via MPP6 was essential for MPP6 to exert its redundancy with RRP6. However, at least for the decay of our identified exosome substrates, MTR4 recruitment by MPP6 was not functionally equivalent to recruitment by RRP6. Genome-wide classification of substrates based on their sensitivity to each exosome component revealed that MPP6 deals with a specific range of substrates and highlights the importance of MTR4 for their decay. Considering recent findings of competitive binding to the exosome between auxiliary complexes, our results suggest that the MPP6-incorporated MTR4-exosome complex is one of the multiple alternative complexes rather than the prevailing one.
... Among these, the zinc-finger protein ZCCHC8 and the RNA-recognition motif (RRM)-containing protein RBM7 were discovered as prominent MTR4-interacting factors in a protein-protein interaction profiling study (Lubas et al., 2011). The resulting MTR4-ZCCHC8-RBM7 trimer, coined the nuclear exosome targeting (NEXT) complex, is now known to target a variety of non-coding RNAs to the nuclear exosome, including promoter upstream transcripts (PROMPTs) (Lubas et al., 2011), enhancer RNAs (eRNAs) (Meola et al., 2016), the 3 0 extended products of sn(o)RNAs (Hrossova et al., 2015;Lubas et al., 2015), telomerase RNA (Tseng et al., 2015), and ncRNAs involved in the antibacterial immune response (Imamura et al., 2018). Overall, the NEXT complex has emerged as a major exosome adaptor for the degradation of non-functional transcripts arising from spurious transcription (Garland and Jensen, 2020). ...
In mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting (NEXT) complex plays a central role in directing non-functional transcripts to exosome-mediated degradation, but the structural and molecular mechanisms remain enigmatic. Here, we elucidated the architecture of the human NEXT complex, showing that it exists as a dimer of MTR4-ZCCHC8-RBM7 heterotrimers. Dimerization preconfigures the major MTR4-binding region of ZCCHC8 and arranges the two MTR4 helicases opposite to each other, with each protomer able to function on many types of RNAs. In the inactive state of the complex, the 3′ end of an RNA substrate is enclosed in the MTR4 helicase channel by a ZCCHC8 C-terminal gatekeeping domain. The architecture of a NEXT-exosome assembly points to the molecular and regulatory mechanisms with which the NEXT complex guides RNA substrates to the exosome.
... To address a possible role of nuclear RNA decay pathways in the control of TE RNAs, we utilized mouse ESC lines disrupted for ZCCHC8 (NEXT) or ZFC3H1 (PAXT) expression through CRISPR-Cas9 engineering ( Figure 1A). The generation of Zfc3h1 −/− cell lines was previously described (Garland et al., 2019), and equivalent Zcchc8 −/− cells were isolated from single-cell clones ( Figure S1A), displaying strong co-depletion of the NEXT factor RBM7 (Hrossova et al., 2015;Mure et al., 2018;Tseng et al., 2015) (Figure 1B), while other associated factors remained unaffected ( Figure S1B). Both Zcchc8 −/− and Zfc3h1 −/− cells were viable under 2i+LIF growth conditions, maintaining cultures at a level of so-called ground-state pluripotency (Ying et al., 2008) and showing normal mRNA expression of stem-cell-specific transcription factors OCT4 and SOX2 ( Figure S1C). ...
... As RBM7 stability is compromised in Zcchc8 −/− cells, ( Figure 1B), we generated Rbm7 −/− cell lines to distinguish the contribution of either NEXT component to the HUSH interaction. Contrary to ZCCHC8 depletion, RBM7 depletion does not affect ZCCHC8 stability (Hrossova et al., 2015;Lubas et al., 2011), which was recapitulated in the Rbm7 −/− cell line ( Figure 3A). We also generated double Zcchc8 −/− ; Zfc3h1 −/− cell lines to determine whether the HUSH interaction is solely mediated by MTR4, which is central for both NEXT and PAXT. ...
Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous “nuclear exosome targeting” (NEXT) and “human silencing hub” (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.
... While our experiments generally detected few proteomic changes, the temporal resolution achieved by the AID system served to group these into immediate or delayed responses to the respective factor depletion. For example, co-depletion events followed the depletion of target protein levels in a timely, consequential manner and were often found to be driven by the loss of a known interaction partner, as exemplified by the destabilization of RBM7 in ZCCHC8 depletions, which disarms the stable trimeric NEXT complex (70)(71)(72). Likewise, the stability of RNA exosome components may also be impacted by the interaction of individual subunits (66). ...
Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3′ end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways.
... Conversely, the nuclear-exosome-targeting (NEXT) complex and the pA-tail-exosome-targeting (PAXT) connection are localized in the nucleoplasm [30,33]. In addition to MTR4, the NEXT complex consists of the Zn-knuckle protein, ZCCHC8 and the RNA binding protein, RBM7 [30,34], which facilitate exosome decay of short immature and mainly unadenylated RNAs [30,33,35,36]. In the case of PAXT, MTR4 associates tightly with the zinc-finger protein, ZFC3H1, which in turn mediates contact to other RNAbinding proteins, including the nuclear polyA-binding protein PABPN1, to mainly target polyadenylated RNA [33,[37][38][39][40][41]. ...
Many long noncoding RNAs (lncRNAs) are localized in the nucleus and play important roles in various biological processes, including cell proliferation, differentiation and antiviral response. Yet, it remains unclear how some nuclear lncRNAs are turned over. Here we show that the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) controls expression levels of NEAT1v2, a lncRNA involved in the formation of nuclear paraspeckles. hnRNPH1 associates, in an RNA-independent manner, with the RNA helicase MTR4/MTREX, an essential co-factor of the nuclear ribonucleolytic RNA exosome. hnRNPH1 localizes in nuclear speckles and depletion of hnRNPH1 enhances NEAT1v2-mediated expression of the IL8 mRNA, encoding a cytokine involved in the innate immune response. Taken together, our results indicate that the hnRNPH1-MTR4 linkage regulates IL8 expression through the degradation of NEAT1v2 RNA.
... ZCCHC8 binds with two proteins: hMTR4, an RNA helicase, and RBM7, an RNA binding protein, to form a nuclear exosome targeting complex (NEXT complex) [174]. The NEXT complex directs a large scale of non-coding RNA for the nuclear exosome degradation, such as lncRNAs, non-coding promoterupstream transcripts, 3ʹ-extended products of histone and small nuclear RNA transcription [174,178,179]. Taken the zinc-knuckle domain of ZCCHC8 and the similarity between the NEXT complex and the TRAMP complex into consideration, both ZCCHC8 and RBM7 might act to recognize and target RNAs for degradation. ...
The zinc finger CCHC-type (ZCCHC) superfamily proteins, characterized with the consensus sequence C-X2-C-X4-H-X4-C, are accepted to have high-affinity binding to single-stranded nucleic acids, especially single-stranded RNAs. In human beings 25 ZCCHC proteins have been annotated in the HGNC database. Of interest is that among the family, most members are involved in the multiple steps of RNA metabolism. In this review, we focus on the diverged roles of human ZCCHC proteins on RNA transcription, biogenesis, splicing, as well as translation and degradation.
... The prime candidate mediating U12 snRNA decay is the nuclear exosome targeting (NEXT) complex. This trimeric cofactor complex consists of MTR4, RBM7 and ZCCHC8 proteins (40) and has been shown to target snRNAs for nuclear exosome-mediated decay (41). To test if the NEXT complex is involved in the decay of 84C>U U12 snRNA, we knocked down MTR4 and RBM7 in RNU12 +/+ and RNU12 84T/84T cells (Supplementary Figure S5). ...
Disruption of minor spliceosome functions underlies several genetic diseases with mutations in the minor spliceosome-specific small nuclear RNAs (snRNAs) and proteins. Here, we define the molecular outcome of the U12 snRNA mutation (84C>U) resulting in an early-onset form of cerebellar ataxia. To understand the molecular consequences of the U12 snRNA mutation, we created cell lines harboring the 84C>T mutation in the U12 snRNA gene (RNU12). We show that the 84C>U mutation leads to accelerated decay of the snRNA, resulting in significantly reduced steady-state U12 snRNA levels. Additionally, the mutation leads to accumulation of 3'-truncated forms of U12 snRNA, which have undergone the cytoplasmic steps of snRNP biogenesis. Our data suggests that the 84C>U-mutant snRNA is targeted for decay following reimport into the nucleus, and that the U12 snRNA fragments are decay intermediates that result from the stalling of a 3'-to-5' exonuclease. Finally, we show that several other single-nucleotide variants in the 3' stem-loop of U12 snRNA that are segregating in the human population are also highly destabilizing. This suggests that the 3' stem-loop is important for the overall stability of the U12 snRNA and that additional disease-causing mutations are likely to exist in this region.
