Prp8 impacts cryptic but not alternative splicing frequency
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2019; 116 (6): 2193–99
Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.
View details for DOI 10.1073/pnas.1819020116
View details for Web of Science ID 000457731900061
View details for PubMedID 30674666
SNRP-27, the C. elegans homolog of the tri-snRNP 27K protein, has a role in 5' splice site positioning in the spliceosome.
RNA (New York, N.Y.)
2018; 24 (10): 1314-1325
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 C. elegans, 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 snrp-27 mutation. The changes in splicing are specific to alternative 5' splice sites. Analysis of new alleles suggests that snrp-27 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.
View details for DOI 10.1261/rna.066878.118
View details for PubMedID 30006499
View details for PubMedCentralID PMC6140464