Prp16 enables efficient splicing of introns with diverse exonic consensus elements in the short-intron rich Cryptococcus neoformans transcriptome

The DEAH box splicing helicase Prp16 in budding yeast governs spliceosomal remodeling from the branching conformation (C complex) to the exon ligation conformation (C* complex). In this study, we examined the genome-wide functions of Prp16 in the short intron-rich genome of the basidiomycete yeast Cryptococcus neoformans. The presence of multiple introns per transcript with intronic features more similar to higher eukaryotes makes it a promising model to study spliceosomal splicing. Using a promoter-shutdown conditional Prp16 knockdown strain, we uncovered its genome-wide but substrate-specific roles in C. neoformans splicing. The splicing functions of Prp16 are dependent on its helicase motif I and motif II that are conserved motifs for helicase activity. A small subset of introns spliced independent of Prp16 activity, were investigated to discover that exonic sequences at the 5 splice site (5SS) and 3 splice site (3SS) with stronger affinity for U5 loop 1 as a common feature in these introns. Furthermore, short (60-100nts) and ultra-short introns (<60nts) prevalent in the C. neoformans transcriptome were more sensitive to Prp16 knockdown than longer introns, indicating Prp16 is required for the efficient splicing of short and ultra-short introns. We propose that stronger U5 snRNA-pre-mRNA interactions enable the efficient transition of the spliceosome from the first to the second catalytic confirmation in Prp16 knockdown, particularly for short introns and introns with suboptimal features. This study provides insights into the fine-tuning spliceosomal helicase functions with variations in cis-element features.