Prp8 impacts cryptic but not alternative splicing frequency

Mayerle, Megan; Yitiz, Samira; Soulette, Cameron M.; Rogel, Lucero E; Ramírez, Andrea Corona; Ragle, James Matthew; Katzman, Sol; Guthrie, Christine; Zahler, Alan M.

doi:10.1073/pnas.1819020116

Cited by 14 publications

(30 citation statements)

References 34 publications

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“…Mutations in the core splicing machinery can also lead to derepression of cryptic splice sites and exons, as shown by analysis of cryptic 3 0 splice sites induced by cancer-associated SF3B1 mutations (Darman et al, 2015;DeBoever et al, 2015). Interestingly, a Caenorhabditis elegans genetic screen recently identified alleles of the core spliceosome component Prp8 that specifically alter cryptic splicing frequency (Mayerle et al, 2019). Notably, alterations in the accuracy of splice site recognition can lead to substantial transcriptome heterogeneity in cancer, with consequences for the generation of neoantigens of possible relevance in immunotherapy (Kahles et al, 2018; https://genome.…”

Section: Reviewmentioning

confidence: 99%

Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution

2019

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High-throughput sequencing-based methods and their applications in the study of transcriptomes have revolutionized our understanding of alternative splicing. Networks of functionally coordinated and biologically important alternative splicing events continue to be discovered in an ever-increasing diversity of cell types in the context of physiologically normal and disease states. These studies have been complemented by efforts directed at defining sequence codes governing splicing and their cognate trans-acting factors, which have illuminated important combinatorial principles of regulation. Additional studies have revealed critical roles of position-dependent, multivalent protein-RNA interactions that direct splicing outcomes. Investigations of evolutionary changes in RNA binding proteins, splice variants, and associated cis elements have further shed light on the emergence, mechanisms, and functions of splicing networks. Progress in these areas has emphasized the need for a coordinated, community-based effort to systematically address the functions of individual splice variants associated with normal and disease biology.

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Section: Reviewmentioning

confidence: 99%

Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution

2019

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“…Splice signal recognition is a highly efficient process and errors in splice signal recognition including splicing from unannotated splice sites (cryptic splicing) often leads to diseases (2,3). How the yeast mature spliceosome prevents incorporation of a sub-optimal/cryptic splice site is currently being investigated (4,5). However, the mechanism of efficient and faithful recognition of the enormous combinatorial library of splice signals in the mammalian transcriptome by the early spliceosomal components is still not fully understood.…”

Section: Introductionmentioning

confidence: 99%

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code

Saha

Fernandez

Biswas

et al. 2018

Preprint

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For splicing of a metazoan pre-mRNA, the four major splice signals -5′ and 3′ splice sites (SS), branch-point site (BS), and a poly-pyrimidine tract (PPT) -are initially bound by splicing factors U1 snRNP, U2AF35, SF1, and U2AF65, respectively, leading up to an early spliceosomal complex, the E-complex. The E-complex consists of additional components and the mechanism of its assembly is unclear. Hence, how splice signals are organized within E-complex defining the exon-intron boundaries remains elusive. Here we present in vitro stepwise reconstitution of an early spliceosome, assembled by cooperative actions of U1 snRNP, SRSF1, SF1, U2AF65, U2AF35, and hnRNP A1, termed here the recognition (R) complex, within which both splice sites are recognized. The R-complex assembly indicates that the SRSF1:pre-mRNA complex initially defines a substrate for U1 snRNP, engaging exons at both ends of an intron. Subsequent 5′SSdependent U1 snRNP binding enables recognition of the remaining splice signals, defining the intron. This R-complex assembly indicates the minimal constituents for intron definition revealing mechanistic principles behind the splice site recognition.

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“…The animals bearing the unc-73(e936) allele are able to live and reproduce through self-fertilization, but are profoundly uncoordinated. Even a modest increase in splicing at the in-frame /UU splice site results in a dramatic phenotypic reversal which is visible at the plate level, making this allele a sensitive assay of perturbations to splice site choice [16,17,19] ). Because those previous screens have identified mutations on residues modelled near the active site of the spliceosome, and those mutations often change global 5' splice site choice, we concluded that a genetic screen using this allele can identify loci which are capable of affecting splice site choice.…”

Section: Resultsmentioning

confidence: 99%

“…This dramatic phenotype is corrected by even a small increase in in-frame splicing, making its suppression screenable. Previously identified dominant mutations that are able to suppress the unc phenotype by altering cryptic splicing in unc- 73(e936) were found in U1snRNA [20] ,] SNRP-27 [16] ; [21] and the largest and most conserved protein in the spliceosome, PRP-8 [17] . The suppressive role these mutations play in this splice site assay provided genetic evidence of a role for these protein residues in 5' splice site choice.…”

Section: Introductionmentioning

confidence: 99%

A Genetic Screen for Suppressors of Cryptic 5’ Splicing inC. elegansReveals Roles for KIN17 and PRCC in Maintaining Both 5’ and 3’ Splice Site Identity

Jm¹,

Osterhoudt²,

Ch³

et al. 2021

Preprint

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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 but not KIN17 changed 5′ splice sites genome-wide, promoting usage of nearby non-consensus sites. We further found that mutations in KIN17 and PRCC changed dozens of 3′ splice sites, promoting non-consensus sites upstream of canonical splice sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.

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Prp8 impacts cryptic but not alternative splicing frequency

Cited by 14 publications

References 34 publications

Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution

Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code

A Genetic Screen for Suppressors of Cryptic 5’ Splicing inC. elegansReveals Roles for KIN17 and PRCC in Maintaining Both 5’ and 3’ Splice Site Identity

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