CS-21-5 - Missense Alleles of the PRP8 and BRR2a Splicing Factors Restore Splicing to Splice-site Mutants

Most plant transcripts contain introns that are removed through a dynamic multistep process orchestrated by five snRNAs and hundreds of proteins that comprise the spliceosome. Intron excision requires precise positioning and assembly of spliceosomal components on the 5’ and 3’ ends and branchpoint of the intron. The 5’ and 3’ splice sites are characterized by conserved AGgt and agGT (intronic residues shown in lowercase) sequences, respectively. The essential G residues in these conserved sequences are sensitive to EMS mutagenesis and are often mutated in alleles recovered from forward-genetic screens. For example, out of sixteen Arabidopsis pex14 alleles recovered from forward-genetic screens for peroxisome dysfunction, eight had mutations in these conserved Gs that disrupted splicing. We used one of these alleles, pex14-6, in a suppression screen for restored splicing, and recovered missense alleles of two essential splicing factor genes, PRP8 and BRR2a. PRP8 and BRR2a are critical to pre-mRNA binding and activation of the spliceosome.  We introduced both splicing factor mutations to a series of pex14 misspliced alleles to probe splice-site fidelity in Arabidopsis. Our prp8 and brr2a mutants restored splicing to pex14 misspliced mutants with mutations in the 5’ exonic G residue but not the 5’ or 3’ intronic G residue.
Our prp8 mutation alters a conserved binding domain for the U5 snRNA, which positions the protein onto pre-mRNA, and our brr2a mutation affects the first helicase domain, which interacts with PRP8 during spliceosome activation. We are using RNA-seq to probe the systemic effects on splicing in our prp8 and brr2a altered specificity mutants compared to previously described missense alleles. This research will elucidate mechanisms of spliceosome intron recognition and the genome-wide consequences of reduced splice-site fidelity. (This research is supported by the NIH.)

Coauthors: Bonnie Bartel – Rice University; Sarah Burkhart – Rice University; Stephanie Xiong – Rice University