Investigating a novel function of snRNP assembly factor Prp24 in regulating H2B monoubiquitylation
A key step in regulation of eukaryotic gene expression is the removal of introns through splicing. While it has been well-established that the majority of splicing occurs co-transcriptionally, many questions remain regarding the coupling of these processes. It has been shown that altering rates of transcription leads to changes in splicing efficiency; however, the mechanism underlying these effects is not well understood. Post-translational modifications (PTMs) of histone proteins are a key regulator of chromatin structure, and several histone PTMs have been associated with transcription and splicing. One such PTM is mono-ubiquitylation of H2B (H2Bub1) at a conserved lysine residue (K123 in S. cerevisiae). A very tight correlation between H2Bub1 levels and active transcription has been demonstrated, and recent studies have linked H2Bub1 to splicing as well. In S. cerevisiae, loss of H2Bub1, either by deletion of the E3 ubiquitin ligase which places the mark (Bre1) or by mutation of the ubiquitylated residue, leads to an increase in intron retention and synthetic growth defects with splicing factor mutants. Additionally, studies in human cells have shown that knockdown of a conserved small nuclear ribonucleoprotein (snRNP) recycling factor, SART3, lead to an increase in H2Bub1. Our investigations have shown this effect is conserved in S. cerevisiae, and is mediated by a highly conserved residue in the first RNA Recognition Motif (RRM) of Prp24, the SART3 ortholog. Interestingly, although RRM1 does not bind snRNAs, mutation of this residue causes significant growth defects and severe temperature sensitivity. Mutations of other RRMs in Prp24, known to affect its ability to bind the snRNA U6, do not affect H2Bub1. These results suggest that Prp24 has a previously uncharacterized role in regulation of H2Bub1. Future experiments will reveal if Prp24’s influence on H2Bub1 is connected to its association with splicing factors. Additional studies will test for genetic and physical interactions between Prp24 and Bre1, as well as deubiquitylating enzymes Ubp8 and Ubp10. Finally, investigation of Prp24’s effects on transcription elongation rate, splicing, and the cell cycle will uncover potential indirect influences on H2Bub1 levels. Together, the results of this investigation will shed light on a novel, likely conserved role of Prp24 in regulating H2Bub1 and contribute new insight into the link between chromatin, transcription, and splicing.