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Involvement of the RNA polymerase II-associated Paf1 complex in transcriptional regulation and 3'-end formation of snoRNAs

Sheldon, Kathryn Elizabeth (2006) Involvement of the RNA polymerase II-associated Paf1 complex in transcriptional regulation and 3'-end formation of snoRNAs. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Sheldon, Kathryn, kathryn.kumer@pitt.eduKEKUMER
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairArndt, Karenarndt@pitt.eduARNDT
Committee CoChairGrabowski, Paulapag4@pitt.eduPAG4
Committee MemberChapman, Deborahdlc7@pitt.eduDLC7
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.eduJBRODSKY
Committee MemberSchmidt, Martinmcs2@pitt.eduMCS2
Date: 20 March 2006
Date Type: Completion
Defense Date: 18 November 2005
Approval Date: 20 March 2006
Submission Date: 8 December 2005
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: 3'-end formation; RNA polymerase II; the Paf1 complex; transcription
Other ID:, etd-12082005-171750
Date Deposited: 10 Nov 2011 20:09
Last Modified: 15 Nov 2016 13:53


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