Structure and regulation of the nuclear exosome targeting complex guides RNA substrates to the exosome.
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ABSTRACT:
SUBMITTER: Gerlach P
PROVIDER: S-EPMC9278407 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
| S-EPMC6124691 | biostudies-literature
Project description:The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared exosome complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel in vitro. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear complex. Many exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic exosome substrates can be distinguished in vivo.
| S-EPMC5389853 | biostudies-literature
Project description:The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.
| S-EPMC4942675 | biostudies-literature
Project description:The RNA-processing exosome contains ribonucleases that degrade aberrant RNAs in archael and eukaryotic cells. In Saccharomyces cerevisiae, the nuclear/nucleolar 3'-5' exoribonuclease Rrp6 distinguishes the nuclear exosome from the cytoplasmic exosome. In vivo, the TRAMP complex enhances the ability of the nuclear exosome to destroy some aberrant RNAs. Previous reports showed that purified TRAMP enhanced RNA degradation by the nuclear exosome in vitro. However, the exoribonucleolytic component(s) of the nuclear exosome enhanced by TRAMP remain unidentified. We show that TRAMP does not significantly enhance RNA degradation by purified exosomes lacking Rrp6 in vitro, suggesting that TRAMP activation experiments with nuclear exosome preparations reflect, in part, effects on the activity of Rrp6. Consistent with this, we show that incubation of purified TRAMP with recombinant Rrp6 results in a 10-fold enhancement of the rate of RNA degradation. This increased activity results from enhancement of the hydrolytic activity of Rrp6 because TRAMP cannot enhance the activity of an Rrp6 mutant lacking a key amino acid side chain in its active site. We observed no ATP or polyadenylation dependence for the enhancement of Rrp6 activity by TRAMP, suggesting that neither the poly(A) polymerase activity of Trf4 nor the helicase activity of Mtr4 plays a role in the enhancement. These findings identify TRAMP as an exosome-independent enhancer of Rrp6 activity.
| S-EPMC2823493 | biostudies-literature
Project description:The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome.
| S-EPMC4310248 | biostudies-literature
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) followed by high-throughput sequencing of RBM7-associated transcripts. Note: these data relate to Figure 1, 2, 3, 4, 5 and 6 in Lubas, Andersen et al., Cell Reports 2014 RBM7-associated transcripts
2014-12-03 | E-GEOD-63791 | biostudies-arrayexpress
Project description:The nuclear exosome targeting complex (NEXT) directs a major 3'-5' exonuclease, the RNA exosome, for degradation of nuclear noncoding (nc) RNAs. We identified the RNA-binding component of the NEXT complex, RBM7, as a substrate of p38(MAPK)/MK2-mediated phosphorylation at residue S136. As a result of this phosphorylation, RBM7 displays a strongly decreased RNA-binding capacity, while inhibition of p38(MAPK) or mutation of S136A in RBM7 increases its RNA association. Interestingly, promoter-upstream transcripts (PROMPTs), such as proRBM39, proEXT1, proDNAJB4, accumulated upon stress stimulation in a p38(MAPK)/MK2-dependent manner, a process inhibited by overexpression of RBM7(S136A). While there are no stress-dependent changes in RNA-polymerase II (RNAPII) occupation of PROMPT regions representing unchanged transcription, stability of PROMPTs is increased. Hence, we propose that phosphorylation of RBM7 by the p38(MAPK)/MK2 axis increases nuclear ncRNA stability by blocking their RBM7-binding and subsequent RNA exosome targeting to allow stress-dependent modulations of the noncoding transcriptome.
| S-EPMC4338353 | biostudies-literature
Project description:Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.
| S-EPMC3923317 | biostudies-literature
Project description:BACKGROUND:The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS:To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS:In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.
| S-EPMC7667871 | biostudies-literature
Project description:Abstract: RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3′ to 5′ ribonucleolytic RNA exosome. Here, we determined cryo-electron microscopy structures of human Nuclear Exosome Targeting (NEXT) complexes bound to RNA, that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 engaging sequences upstream of the 3′ end that is bound by MTR4. ZCCHC8 obscures MTR4 surfaces that are important for RNA binding and extrusion as well as surfaces important for MPP6-dependent recruitment and docking onto the RNA exosome core, features of which might contribute to coordinating RNA translocation and extrusion from the helicase to exosome recruitment and decay.
2022-06-09 | GSE185374 | GEO