RESUMEN
RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3' splice site (3'ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3' splicing in introns with pairs of 3' splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3'ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3'ss usage in the germline for â¼200 events; many of the somatic SACY-1 alternative 3' splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3' splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3'ss.
Asunto(s)
Sitios de Empalme de ARN , Empalmosomas , Humanos , Sitios de Empalme de ARN/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Empalme del ARN , Empalme Alternativo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ADN Helicasas/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismoRESUMEN
The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5' splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5'ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5'ss identified by U1snRNA for entry into the catalytic core.
Asunto(s)
Sitios de Empalme de ARN , Factores de Empalme de ARN , Empalmosomas , Animales , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Mutación , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Factores de Empalme de ARN/genéticaRESUMEN
Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5' GU and ending with 3' AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5' splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5' splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5' splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5' splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.
