Genetic Studies of Co-transcriptional pre-mRNA Splicing Regulation
During eukaryotic gene expression, non-coding intron sequences are removed from the protein-coding exon sequences of pre-messenger RNA (pre-mRNA) by the process of splicing. Splicing is carried out by the spliceosome, a macromolecular ribozyme that assembles de novo on every intron. It has been established that the spliceosome assembles co-transcriptionally while the nascent pre-mRNA is attached to the elongating RNA polymerase II (RNAP II). However, mechanisms that enforce the coupling of splicing to transcription and the chromatin landscape remain poorly understood.
Here, I describe our work to gain further insight into the regulation of co-transcriptional pre-mRNA splicing by employing genetic and molecular biology approaches. Using the traditional splicing model organism Saccharomyces cerevisiae, we interrogated the mechanism of a rapid splicing block at the ribosomal protein genes (RPGs) that occurs in response to amino acid starvation stress. By creating chimeric gene constructs, we show that an RPG promoter sequence element, and not the intron sequence, is both necessary and sufficient to confer a transcript-specific splicing response. This study reveals a mechanism whereby in response in stress RNAP II becomes de-enriched near the 3´splice site and early splicing factor recruitment is delayed, consistent with an environmentally-responsive splicing-dependent polymerase checkpoint, and underscoring the highly coordinated nature of splicing and transcription.
We further expanded our studies of splicing regulation into Schizosaccharomyces pombe, a emerging model organism that shares many splicing features with mammalian systems, such as abundant non-consensus splice sites, multi-intron containing genes, and conserved trans-acting splicing factors. In our work with the S. pombe system, we developed techniques for probing splicing factor association with chromatin. Starting from a genome-wide genetic screen, we characterized interactions between the spliceosome and Swr1 complex, which deposits the highly conserved histone variant H2A.Z. We identified H2A.Z’s requirement in efficient splicing at introns with weak splice sites, supporting a growing hypothesis of chromatin-dependent splicing checkpoints. Taken together, these studies reveal an unappreciated role of the promoter and its proximal nucleosome composition in transcript-specific pre-mRNA splicing regulation. We expect these results to provide foundational insights into the interconnectedness of gene expression processes and have important implications in human disease.