Transcription elongation is an important regulatory step in the RNA polymerase II transcription cycle. Previous work from our laboratory and others strongly suggests that the Paf1 complex interacts with and regulates the activity of RNA polymerase II during transcription elongation. Affinity purification of Paf1 revealed that it exists in a complex in vivo with Ctr9, Rtf1, Cdc73 and Leo1 called the Paf1 complex. To further investigate the function of the Paf1 complex in vivo, we used a genetic approach to study the component Rtf1 and microarray analyses to identify Ctr9-regulated genes. Null mutations in RTF1 confer two phenotypes associated with defective transcription elongation, sensitivity to base analogs and the Spt- phenotype. To identify novel mutations in RTF1, we performed a genetic screen for rtf1 mutations that confer conditional mutant phenotypes. We identified three new rtf1 missense mutations. To identify proteins that functionally interact with the Paf1 complex, we performed a screen for multicopy suppressors of the rtf1 mutations. This screen identified NAB3 as a suppressor of rtf1. Nab3 interacts with the RNA polymerase II-associated hnRNP Nrd1. Together, Nrd1 and Nab3 are required for efficient 3'-end formation of certain nonpolyadenylated RNA polymerase II transcripts, including snoRNAs. Using assays to detect transcriptional readthrough of snoRNA genes, we have shown that deletion of certain Paf1 complex members causes strong defects in snoRNA 3'-end formation. By chromatin immunoprecipitation (ChIP) analysis, we have found that the Paf1 complex and Nab3 associate with snoRNA genes in vivo. In the second approach, expression analyses revealed a role for the Paf1 complex in directly regulating the expression of certain genes that are not transcribed during growth in rich media. ChIP experiments indicated that the Paf1 complex associates along the length of ARG1 and with the promoter region of SER3. Together, our results reinforce the idea that the Paf1 complex is involved in the production of mRNAs and provide evidence that the Paf1 complex also participates in snoRNA transcription and 3'-end formation during RNA polymerase II transcription.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12082005-171750 |
| Date | 20 March 2006 |
| Creators | Sheldon, Kathryn Elizabeth |
| Contributors | Jeffrey Brodsky, Deborah Chapman, Martin Schmidt, Paula Grabowski, Karen Arndt |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12082005-171750/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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