Title: Regulation of alternative splicing and its connections to cancer
Description: This thesis presents two separate pieces of work pertaining to pre-mRNA splicing in mammalian cells. The first piece, as the main research project of the thesis, consists of two related parts. The first part identified the regulators of the alternative splicing of the PKM gene in cancer cells while the second part elucidates the molecular mechanism of how this mutually exclusive alternative splicing is regulated. The second piece investigates the molecular mechanism of how SRp38 functions as a splicing activator when phosphorylated. Cancer cells uniformly alter key aspects of their metabolism, including their glucose usage. In contrast to quiescent cells, which use most of their glucose for oxidative phosphorylation when oxygen is present, under the same conditions, most of the glucose consumed by cancer cells is converted to lactate. This phenomenon is known as aerobic glycolysis, and is critical for cancer cell growth. The pyruvate kinase isoform expressed by the cell is a key determinant of glucose usage.

Pyruvate kinase in most tissues is produced from the PKM gene, which is alternatively spliced to produce the PKM1 or PKM2 isoforms, which contain exons 9 or 10 respectively. Adult tissues, such as skeletal muscle and brain, express predominantly the PKM1 isoform, which is universally reverted to the embryonic PKM2 isoform in cancer cells. PKM2 expression promotes aerobic glycolysis. In Chapter 3, I describe a mechanism by which cancer cells promote switching to PKM2. We show that PKM exon 9 is flanked by binding sites for the RNA-binding proteins hnRNP A1/A2 and PTB. These proteins bind to exon 9 and repress its inclusion in the mRNA, resulting in PKM2 production. Additionally, we show that hnRNP A1/A2 and PTB are all overexpressed in cancers in a way that precisely correlates with the expression of PKM2. Finally, we show that the oncogenic transcription factor c-Myc promotes PKM2 expression by transcriptionally upregulating the genes encoding hnRNP A1/A2 and PTB. In Chapter 4, I provide additional insights into how PKM AS is regulated and a novel discovery that general splicing repressors can repress either one of the two mutually exclusive exons at different expression levels, through protein-protein interactions of these proteins bound on different sets of binding sites on and flanking each.

First, using a splicing minigene construct that recapitulates PKM splicing in HeLa cells, we identified additional PTB and hnRNP A1/ A2 ISSs in intron 9 necessary for full exclusion of exon 9. More importantly, we found two ESSs in exon 9, absent from exon 10, that match the hnRNP A1 consensus, and which are critical for exon 9 exclusion. We show that these ESSs function cooperatively to facilitate hnRNP A1 binding to an intronic splicing silencer in intron 9 described in Chapter 3. I also elucidated the mechanism of how exon 10 is excluded when exon 9 is derepressed and show that hnRNP A1 and PTB, when their protein levels are reduced, release the inhibition of exon 9 but repress exon 10 inclusion, through binding sites present in introns 9 and 10. This mechanism, coupled with nonsense mediated decay, function to prevent the appearance of PKM mRNA containing both exon 9 and exon 10. In the second piece of work, presented in Chapter 5, I, based on the findings from a previous post doctor that SRp38 functions as a sequence-specific splicing activator, showed that SRp38 promotes spliceosomal complex A formation.

I examined the mechanism of spliceosomal A complex formation and found that SRp38 promotes the recruitment of U1 and U2 snRNPs to splicing substrates that contain high-affinity SRp38 binding sites.