Regulation of RNA Synthesis and Decay Via the C-terminal Domain of Pol II PDF Download

Author: Sandra Chang Tseng
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Languages : en
Pages : 213

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Book Description
RNA expression can be thought of as a sum of the products of RNA synthesis and decay: on the one hand, cells are transcribing the genes required for a given situation, while on the other, RNA degradation machinery is constantly acting on normal as well as aberrantly synthesized RNA. The kinases which phosphorylate the C-terminal domain (CTD) of RNA Polymerase II (Pol II) are canonically associated with being on the additive scale of the balancing act between RNA synthesis and decay. One such kinase, and the focus of my thesis, Kin28/CDK7 activates and promotes transcription by phosphorylating serine residues on the CTD. I begin my thesis with an introduction into our current understanding of how transcription is regulated via post-translational modification of the CTD, and also present the open questions, which this thesis seeks to address, on the role of Kin28/CDK7 in mediating gene expression (Chapter 1). In Chapter 2, I describe the development of a chemical genetic approach to covalently inhibit Kin28 in budding yeast. The strategy and lessons learned therein can be generally applied to covalently inhibit kinases in other model organisms. Combined with transcriptomic sequencing, I show that Kin28 is required for synthesis of nearly all annotated yeast mRNAs, and demonstrate that previously conflicting views on the matter can be explained by the phenomenon known as "RNA buffering," which is the stabilization of the steady-state level of RNAs during insults to transcription or decay. Also discussed in this chapter is our novel discovery that Kin28 plays a role in advancing Pol II from the initiation into the elongation phase of transcription. In Chapter 3, I investigate determinants of RNA buffering in a Kin28-inhibited regime, and reveal that the relative binding of the RNA binding proteins Nab2 and Ski2 can predict mRNA stability. Furthermore, I show inhibition of Kin28 rapidly induces the formation P bodies and perturbs translation. From my analyses, I present a compendium of the mRNAs that are unstable, buffered, translated, and or poorly translated in situations of transcriptional crisis, a state I show is distinct from that of the canonical stress response. In Chapter 4, I study RNA buffering from the "other side" of the gene expression equation by exploring the combined effect of deleting a component of the RNA decay machinery (rai1::URA3) and inhibition of Kin28. Despite being on opposite sides of the scale of gene expression, both defects in decay (rai1::URA3) and synthesis (inhibition of Kin28) result in similar outcomes, such as the formation of P bodies and defects to translation, suggesting translation as a key regulatory node for the crosstalk between RNA synthesis and decay. Finally, in Chapter 5, I comment on the potential of Kin28/CDK7-based therapies for the treatment of disease, and how studies in yeast can inform on the mechanism of action of such therapies. Appendix I describes my efforts into characterizing the role of CTD methylation on gene expression. In sum, this work has significantly contributed to our understanding of the myriad ways Kin28/CDK7 functions in regulating gene expression beyond its canonical roles in promoting RNA synthesis.