Studies on mammalian pre-mRNA splicing : connections to transcription and cancer

This thesis presents two separate pieces of work pertaining to pre-mRNA splicing in mammalian cells. The first part examines the regulation of the alternative splicing of the PKM gene in cancer cells, while the second part investigates the physical connections between the transcriptional apparatus and splicing factors. 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 to produce the PKM1 or PKM2 isoforms, which contain exons 9 or 10 respectively. Adult tissues 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 the second part of my work, presented in Chapter 5, I examine the coupling of transcription and splicing. The RNA polymerase II C-terminal domain (CTD) plays an important role in ensuring that pre-mRNA transcripts are efficiently spliced, most likely through interactions between splicing factors and the CTD. We have established a biochemical complementation system that has facilitated the identification of a splicing factor that binds to the CTD. Surprisingly, purification of the factor revealed it to be a complex containing U2AF65 and the Prp19 complex, two central splicing factors that had not previously been shown to interact. This complex is functional: I present evidence that the two factors can only activate splicing of the IgMA3 pre-mRNA when they are engaged in a complex. I go on to show that U2AF65 binds directly to the CTD, and this interaction stimulates the RNA binding of U2AF65