RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3' end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3' end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation. We further provide evidence that Thr 4 functions in histone mRNA 3' end formation (presented mostly in chapter 2 of this thesis). Chapter 3 mainly describes the studies regarding Ser 2, Ser 5, and Ser 7. Based on the DT40-Rpb1 cell line, I created stable cell lines expressing an Rpb1 carrying a CTD with Ser 2, Ser 5, or Ser 7 mutated to alanine, and investigated the phenotypes of these cells. We found that cells expressing an Rpb1 with S2A or S5A mutation were defective in transcription and RNA processing. Contrary to previous findings, we found Ser 7 was not involved in snRNA expression and 3' end processing. In fact, no phenotypes associated with Ser 7 mutation were detected by our measurements. Extending previous Thr 4 studies, we showed in vitro and in vivo that Fcp1 dephosphorylated Thr 4. Finally, Chapter 4 describes what we have found the functions of CTD Tyr 1. Using the DT40-Rpb1 cells, I created stable cell lines expressing an Rpb1 with all Tyr residues mutated to phenylalanine (Phe). We found these cells were inviable, and the mutant Rpb1-Y1F was degraded to a CTD-less protein. Interestingly, the instability of Rpb1-Y1F was restored by reintroduction of one Tyr residue at the last heptad repeat. Further analysis provided evidence showing the involvement of Tyr phosphorylation in preventing Rpb1 from degradation by the 20S proteasome. Next, using ChIP assay, we showed Tyr phosphorylation was detected mostly at promoters, indicating a function of Tyr phosphorylation in transcription initiation. Indeed, transcription initiation defects were uncovered by assessing the recruitment of general transcription factors in cells with Y1F mutation. Extending this, we found an accumulation of upstream antisense RNAs in about one hundred reference genes by RNA-Seq analysis.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8GH9H96 |
| Date | January 2013 |
| Creators | Hsin, Jing-Ping |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
Page generated in 0.0019 seconds