SNRP-27, the C. elegans homolog of the tri-snRNP 27K protein, has a role in 5′ splice site positioning in the spliceosome

Abstract

The tri-snRNP 27K protein is a component of the human U4/U6-U5 tri-snRNP and contains an N-terminal phosphorylated RS domain. In a forward genetic screen in , we previously identified a dominant mutation, M141T, in the highly-conserved C-terminal region of this protein. The mutant allele promotes changes in cryptic 5' splice site choice. To better understand the function of this poorly characterized splicing factor, we performed high-throughput mRNA sequencing analysis on worms containing this dominant mutation. Comparison of alternative splice site usage between the mutant and wild-type strains led to the identification of 26 native genes whose splicing changes in the presence of the mutation. The changes in splicing are specific to alternative 5' splice sites. Analysis of new alleles suggests that is an essential gene for worm viability. We performed a novel directed-mutation experiment in which we used the CRISPR-cas9 system to randomly generate mutations specifically at M141 of SNRP-27. We identified eight amino acid substitutions at this position that are viable, and three that are homozygous lethal. All viable substitutions at M141 led to varying degrees of changes in alternative 5' splicing of native targets. We hypothesize a role for this SR-related factor in maintaining the position of the 5' splice site as U1snRNA trades interactions at the 5' end of the intron with U6snRNA and PRP8 as the catalytic site is assembled.

Prp8 impacts cryptic but not alternative splicing frequency

“…Up 3′ SS use strain 1,2, % that the changes in 3′ SS selection may be a consequence of altered interactions between Prp8 and the pre-mRNA branchpoint. We are confident in our analysis pipeline as this methodology previously yielded 26 high-confidence alternative splicing events for the snrp-27(az26) allele (24). While the snrp-27 and two prp-8 alleles have almost identical effects on unc-73(e936) cryptic 5′ SS choice, our sequencing analysis indicates that they can have differential effects on alternative splicing, suggesting that they may operate at distinct stages of splicing.…”

“…CRISPR Mutagenesis. CRISPR Cas9-directed mutagenesis to generate az29, az50, and T524 and G654 randomization alleles was performed using a co-CRISPR protocol as described (24). prp-8 repair oligos are described in SI Appendix, Table S3.…”

“…prp-8 repair oligos are described in SI Appendix, Table S3. Restriction digests of PCR products were used to track mutations (24,26). , Table S4.…”

“…RNA-Seq Sample Preparation and Analysis. RNA was isolated for library construction from mixed stage populations of C. elegans (September 2017 preps) or populations enriched for young adults (May 2016 preps) as described (24). FASTQ files and processed .gtf files are in Gene Expression Omnibus accession no.…”

“…The sum of significant changes for an event across all pairwise comparisons is the pairSum. The delta PSI cutoff of 0.20 was chosen because of our previous experience and the issue of false flags that require individual inspection (Table 3) (24). A minimum change in alternative SS usage of 20% allowed us to separate true changes in alternative splicing.…”