Defining the crosstalk between the transcription and splicing machineries

Abstract

Production of mammalian messenger RNAs (mRNAs) often requires RNA Polymerase II (RNAPII) to transcribe tens of kilobases and coordinate the co-transcriptional removal of multiple introns by the spliceosome. The expansion of AT-rich introns across evolution introduces significant challenges to RNA biogenesis, raising interest in understanding how the transcription and splicing machineries have adapted to prevent the production of aberrant transcripts in mammalian cells. To help overcome this burden, the spliceosome has been reported to stimulate transcription, however, the mechanisms underlying the crosstalk between the transcription and splicing machineries remain unclear.

The work in this thesis utilizes a repertoire of next-generation sequencing techniques across multiple biological systems to advance our understanding of co-transcriptional splicing in mammals. In Chapter 2, I show that introns are removed rapidly by the spliceosome, often shortly after the intron is fully transcribed by RNAPII, and that intron removal is largely coordinated across individual transcripts. Furthermore, I demonstrate that RNAPII does not pause at intron boundaries. This result refutes models suggesting that RNAPII elongation is altered at intron boundaries in response to splicing and confirms that mammalian spliceosomes can act rapidly.

Next, I describe the impact of sequence content and the spliceosome on transcription. In Chapter 3, I show that AT-rich sequences such as introns have a negative impact on RNAPII elongation potential. However, I find that a 5’ splice site (SS), the intron start boundary, can alleviate these negative effects. In Chapter 4, I probe RNAPII elongation properties after inhibiting 5’ SS recognition by splicing factor U1 snRNP. I demonstrate that U1 snRNP increases RNAPII elongation rate in introns, thereby reducing the likelihood of premature termination and arrest of RNAPII in these AT-rich sequences. Consequently, I show that U1 snRNP is critical for the synthesis of long genes and propose that U1 snRNP has evolved this stimulatory function to mitigate the risk that long mammalian introns pose to RNA production.

Altogether, this thesis provides key mechanistic insights to our basic understanding of co-transcriptional splicing in mammals. In Chapter 5, I discuss future directions and relate these finding to disease models with reoccurring mutations in the spliceosome.