MOLECULAR ANALYSES OF THE DROSOPHILA RNA POLYMERASE II CARBOXY-TERMINAL DOMAIN
Open Access
- Author:
- Lu, Feiyue
- Graduate Program:
- Molecular, Cellular and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 28, 2019
- Committee Members:
- David Scott Gilmour, Dissertation Advisor/Co-Advisor
David Scott Gilmour, Committee Chair/Co-Chair
Benjamin Franklin Pugh, Committee Member
Melissa Rolls, Committee Member
Scott A Showalter, Outside Member
Joseph C. Reese, Committee Member
Claire Madison Thomas, Committee Member - Keywords:
- RNA Pol II CTD
consensus heptads
divergent motifs
transcription compartment
Drosophila genetics - Abstract:
- The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) is a hub that coordinates transcription, RNA processing, and modulation of chromatin structure. The lengths and sequence complexity of the CTDs roughly scale with evolutionary complexity of species. For example, the yeast CTDs have 26 to 29 repeats, and are comprised almost exclusively of repeating Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (Y1S2P3T4S5P6S7) consensus heptads. The mammalian CTDs have 52 repeats, more than half of which are divergent motifs that deviate from the consensus heptads at one or more residue positions. Metazoan CTDs have well-conserved lengths and sequence compositions, features that have been long thought to be essential for gene expression in higher eukaryotes. However, their contribution to development remains largely untested. Here, I present multiple genetic evidence showing that: (1) not all repeats of the CTD are functionally equivalent for Drosophila development; (2) the highly conserved Drosophila CTD divergent motifs can be replaced with the human counterpart; (3) a CTD composed either solely of consensus heptads, or exclusively of divergent motifs, is sufficient to support fly growth; (4) either having too few repeats or having too many consensus heptads is deleterious. These findings support a model where the function of the CTD is largely dependent on its multivalency. Additionally, it was recently shown that transcription factors, enhancers, RNA and Pol II can form liquid like droplets. The formation of such droplets is dependent on weak, multivalent interactions occurring on low-complexity domains. The CTD, which is a low-complexity domain, has been shown to partition into hydrogels containing other transcription factors and is able to phase separate by itself in vitro. To test if the CTD can enter liquid-like active transcription phases in vivo, we express a fluorescently-tagged wild-type CTD in the Drosophila salivary glands and show that the CTD alone dynamically enters transcription compartments. However, highly consensus CTDs are more prone to localize to extrachromosal foci. This suggests that the increase in sequence complexity of the CTD reverts aberrant behavior caused by excessive valency. In a subsequent attempt to analyze the effects of CTD mutations on Pol II distribution genome-wide, I discovered that mutations in the CTD can lead to reduced amount of Pol II on sites of active transcription. Together, the data in this thesis describe the most thorough investigation of the significance of CTD motifs in a multicellular organism and identify the mistargeting of the CTD as one possible molecular basis for the deficiency of CTD mutants.