UCSF

Splicing of eukaryotic pre-mRNAs often occurs co-transcriptionally; however, the components of the co-transcriptional environment that influence splicing remain incompletely defined. The work presented here discusses the effects of altering histone modifications, in particular H2B ubiquitination, and RNA Polymerase II (RNAPII) elongation rate on splicing in Saccharomyces cerevisiae.

To begin our exploration into the effect of the co-transcriptional environment on splicing, we interrogated the genetic, functional, and biochemical interactions of Npl3, an SR-like protein in S. cerevisiae that promotes co-transcriptional spliceosome assembly. Among many chromatin and transcription factors, we found that Npl3 genetically interacts with the histone H2B ubiquitination machinery and went on to show that strains lacking H2B ubiquitination have a mild splicing defect (Chapter 1). We subsequently capitalized on the observation that H2B ubiquitination-dependent processes are sensitive to high temperatures, and observed splicing defects upon inhibition of H2B ubiquitination or related modifications, H3K4 and H3K36 methylation, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analyses on intron-containing genes indicated that H2B ubiquitination stimulates recruitment of the early splicing factors onto nascent RNAs (Chapter 2).

It is challenging to nail down the precise mechanisms by which splicing responds to histone modifications - direct physical interactions between the modification and the spliceosome would offer locus- and modification-specific recruitment to promote splicing efficiency, but an additional variable, RNAPII elongation rate, had been shown to be a key player in metazoans. We therefore interrogated the effect on splicing of altering RNAPII elongation rate in S.cerevisiae, using an allelic series of point mutants that cause fast and slow elongation in vitro. Our splicing microarray experiments revealed that RNAPII speed and splicing efficiency are anti-correlated: at many genes, increased elongation rate caused decreased splicing efficiency, while decreased elongation rate increased splicing efficiency (Chapters 3 and 4).

Taken together, our data support the growing notion that histone modifications and RNAPII elongation rate exert gene-specific effects and potentially offer multiple locus-specific mechanisms for modulating splicing in a dynamic context.