Dissecting a Role for Protein Arginine Methylation on the Recruitment of Spliceosomal Factors

During the process of eukaryotic gene expression, pre-mRNAs undergo a myriad of processing events including splicing in order to generate a mature mRNA. This mature mRNA is then packaged into a messenger ribonucleoparticle (mRNP) and exported out of the nucleus into the cytoplasm. Co-transcriptional recruitment of RNA processing factors to nascent transcripts is required for the proper formation of export-competent mRNPs. In Saccharomyces cerevisiae , ordered, Co-transcriptional recruitment of spliceosomal subunits facilitates correct processing of mRNAs. Previous studies have shown that the predominant S. cerevisiae protein arginine methyltransferase Hmt1 has a major function in the Co-transcriptional recruitment of pre-mRNA splicing factors. However, we do not know the molecular basis by which Hmt1 modulates the recruitment of these splicing factors.;In the first part of this dissertation, I determined the effects of Hmt1 on the recruitment of early splicing factors across intron-containing genes harboring different splice sites. Our data show increase in the recruitment of the SR-/hnRNP-like protein Npl3 and a concomitant decrease in the U1 snRNP subunit Snp1 across a number of intron-containing genes in cells lacking Hmt1. Furthermore, we provide evidence that protein arginine methylation of Npl3 facilitates proper splicing of the SUS1 intron 1, an intron that harbors a non-consensus 5'-splice site and branch site, but not that of other introns harboring consensus splice sites. Additional biochemical studies show that methylation of Npl3 promotes its dissociation from Mud1, a subunit of the U1 snRNP. Overall, our data implicates a function for Hmt1, via Npl3 methylation, in regulating how the U1 snRNP engages different pre-mRNAs to promote efficient splicing of their introns, especially for those that harbor non-consensus splice sites.;The second part of my thesis describes the discovery of redundancy between the functioning of HMT1 and MUD1 genes. We employed a genetic approach to test whether HMT1 regulates the stability of the interaction between the U1 snRNA and non-canonical pre-mRNA 5'SS. Our results reveal a role for Hmt1 in regulating the growth of cells that harbor either hyperstabilized or hypostabilized U1 snRNA-pre-mRNA duplexes. In addition, we identified a functional redundancy between HMT1 and MUD1 that is evident when the U1 snRNA-pre-mRNA duplex is hyperstabilized. This work provides valuable insight into how Hmt1 promotes splicing when encountered with different splice site combinations that resemble the scenario of different thermodynamic stability in the U1 snRNA-pre-mRNA interaction.