A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype in .

Splicing of pre-mRNA involves two consecutive -esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced reporter gene in , we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3' splice site accompanied by ligation of the 5' and 3' exons and intron removal. In the mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the mutant identified numerous cases of intron retention and a preponderance of altered 3' splice sites, suggesting an influence of PRP18a on 3' splice site selection. The mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.