Structure of an aberrant spliceosome intermediate on its way to disassembly
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ABSTRACT:
PROVIDER: PRJNA986513 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24996075_1.fastq.gz | Fastqsanger.gz | |||
| SRR24996075_2.fastq.gz | Fastqsanger.gz | |||
| SRR24996076_1.fastq.gz | Fastqsanger.gz | |||
| SRR24996076_2.fastq.gz | Fastqsanger.gz | |||
| SRR24996077_1.fastq.gz | Fastqsanger.gz |
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Project description:Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to discard is not available. Here, we report the cryo-EM structure of a B state spliceosome intermediate primed for disassembly. We identify the DEAH-box helicase – G patch protein pair (Gih35-Gpl1) to maintain catalytic dormancy with Gpl1 recognizing a remodeled active site of the spliceosome due to a single-nucleotide insertion at the 3’ end of the 5’ exon. Remodeling is communicated to the spliceosome surface and the Ntr1 complex is recruited. Our data pave the way for a targeted analysis of spliceosome-associated quality control.
2024-06-01 | GSE235589 | GEO
Project description:The spliceosome, a highly dynamic macromolecular assembly, catalyzes the precise removal of introns from pre-mRNAs. Recent studies have provided comprehensive structural insights in the step-wise assembly, catalytic splicing and final disassembly of the spliceosome. However, the molecular details of how the spliceosome recognizes and rejects suboptimal splicing substrates remained unclear. Here, we show cryo-electron microscopy structures of spliceosomal quality control complexes from a thermophilic eukaryote, Chaetomium thermophilum. The spliceosomes, henceforth termed B*Q, are stalled at a catalytically activated state but prior to the first splicing reaction due to an aberrant 5’ splice site conformation. This is recognized by G-patch protein GPATCH1, which is docked onto PRP8-EN and ‑RH domains and has recruited its cognate DHX35 helicase to its U2 snRNA substrate. In B*Q, DHX35 has dissociated the U2/branch site helix, while the disassembly helicase DHX15 is docked close to its U6 RNA 3’ end substrate. Our work thus provides mechanistic insights into the concerted action of two spliceosomal helicases in maintaining splicing fidelity by priming spliceosomes that are bound to aberrant splice substrates for disassembly.
2025-03-18 | GSE287473 | GEO
Project description:A battery of spliceosome-associated proteins has been identified in microRNA (miRNA) biogenesis; however, the underlying mechanisms remain elusive. The intron lariat spliceosome (ILS) complex is highly conserved among eukaryotes and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, ILP1 and NTR1, positively regulate microRNA biogenesis through facilitating transcriptional elongation in Arabidopsis. ILP1 and NTR1 form a stable complex and co-regulate alternative splicing of more than a hundred genes across the genome including the core circadian gene LHY and some pri-miRNAs. Dysfunction in either ILP1 or NTR1 result in reduced RNA polymerase II occupancy at elongated regions of MIR chromatins, without affecting MIR promoter activity, pri-miRNA decay and DCL1 processing. Our results provide insights into the molecular mechanisms of spliceosomal machineries in non-coding RNA regulation.
2019-11-12 | PXD011542 | Pride
Project description:Structural insights into spliceosome fidelity: DHX35-GPATCH1 mediated rejection of aberrant splicing substrates
| PRJNA1213057 | ENA
Project description:We identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3’-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.
2024-02-04 | GSE246283 | GEO
Project description:Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.
2021-05-01 | GSE151141 | GEO
Project description:The studies of spliceosomal interactions are challenging due to their dynamic nature. Here we developed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs) to simultaneously map the spliceosomal binding to human snRNAs and pre-mRNAs. This identified 9 distinct regions on pre-mRNAs, which overlap with position-dependent binding patterns of 15 RBPs. Using spliceosome iCLIP, we additionally identified >50,000 branchpoints (BPs) that have canonical features, unlike those identified by RNA-seq. The iCLIP BPs generally overlap with the computationally predicted BPs, and alternative BPs are associated with extended regions of structurally accessible RNA. We find that the position and strength of BPs defines the binding patterns of SF3 and U2AF complexes, whereas the RNA structure around BPs affects the sensitivity of exons to perturbation of these complexes. Our findings introduce spliceosome iCLIP as a new method for transcriptomic studies of BPs and splicing mechanisms.
2019-08-30 | E-MTAB-6950 | biostudies-arrayexpress
Project description:Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.
2021-05-01 | GSE151140 | GEO
Project description:Pre-mRNA splicing is a crucial regulatory process in eukaryotes, ensuring accurate mRNA maturation and contributing to transcript diversity through alternative splicing (AS). This process is mediated by the spliceosome, a large molecular complex composed of small nuclear RNAs (snRNAs) and proteins. Structural and comparative analysis have showed that splicing factors are highly conserved across eukaryotes, though the extent of their functional conservation remains to be fully elucidated. In Arabidopsis, two homologous genes, PRP43a and PRP43b, encode components involved in spliceosome disassembly. To investigate the roles of these genes, we characterized T-DNA insertion mutants prp43a-1 and prp43b-1. While prp43b-1 homozygous mutants were embryonic lethal, prp43a-1 mutants displayed no significant developmental defects under normal conditions, nor did they exhibit impaired heat tolerance. However, RNA-seq analysis revealed transcriptional changes in prp43a-1 under normal conditions. Gene ontology (GO) analysis indicated and enrichment of stress-related processes among the differentially expressed genes (DEGs), suggesting a role for PRP43a in stress responses. Additionally, differential alternative splicing (DAS) analysis identified numerous PRP43a-dependent intron retention (IR) events, particularly under heat stress, indicating that PRP43a influences AS under environmental conditions. These findings suggest PRP43a as a modulator of splicing and gene expression, with potential roles in stress responses.
2024-12-31 | GSE276909 | GEO