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Influence of Protein Binding and Competition on 7SK Ribonucleoprotein Complex Maintenance and Transition

Totten, Daniel (2018) Influence of Protein Binding and Competition on 7SK Ribonucleoprotein Complex Maintenance and Transition. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

As the first step of gene expression, transcription is tightly regulated to ensure the proper growth, development, and homeostasis of an organism. In metazoans, an extra layer of control, known as promoter proximal pausing, is exerted approximately fifty nucleotides into elongation through the association of negative elongation factors with RNA polymerase II. The cyclin-dependent kinase positive transcription elongation factor b (P-TEFb) phosphorylates both the negative elongation factors and the C-terminal domain of the polymerase to release paused RNA polymerase II into productive elongation. Promoter proximal pausing is a highly pervasive control mechanism, as inhibition of P-TEFb abolishes global transcription. P-TEFb activity is predominantly controlled through sequestration and release from an inhibitory ribonucleoprotein complex (RNP) containing the non-coding small nuclear 7SK RNA (7SK-P-TEFb RNP). Release of P-TEFb from this RNP allows 7SK RNA to bind various heterogeneous nuclear ribonucleoproteins (hnRNPs) to form the 7SK-hnRNP RNPs. The transition between the 7SK-P-TEFb RNP and the 7SK-hnRNP RNPs controls active P-TEFb levels, and thus metazoan transcription. The functional consequence of assembly and transition between the 7SK RNPs is unknown. In this thesis, I investigated the effects of protein binding and competition in establishing, maintaining, and transitioning the respective 7SK RNPs in two studies. In the case of hnRNP A1 and serine-arginine splicing factor 2 (SRSF2) binding to Stem III of 7SK RNA, I found that the proteins differentially restructure the RNA element upon binding, helping to maintain exclusive RNP complex formation at high concentrations. However, the formation of an intermediate complex with low concentrations of both factors helps mediate SRSF2 dissociation from the complex during RNP transition. In my second study, I found that direct phosphorylation of hnRNP K by P-TEFb modulates occupancy of competing proteins on Stem I of 7SK RNA. Furthermore, this post-translational modification may play a downstream role in mediating proper termination at select genes. Together, these studies detail the first biochemical examination of hnRNP-7SK RNA interactions and suggest that 7SK RNP maintenance and transition is controlled by transcription-dependent changes in the local concentrations of RNA-binding factors.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Totten, DanielDCT18@Pitt.eduDCT18
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBerman, AndreaAJB190@pitt.edu
Committee MemberSaunders, Williamwsaund@pitt.edu
Committee MemberWoolford, Johnjw17@andrew.cmu.edu
Committee MemberGrabowski, Paulapag4@pitt.edu
Committee MemberArndt, Karenarndt@pitt.edu
Date: 31 January 2018
Date Type: Publication
Defense Date: 11 October 2017
Approval Date: 31 January 2018
Submission Date: 13 November 2017
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 170
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: 7SK RNA, hnRNP A1, hnRNP K, P-TEFb regulation, metazoan transcription regulation
Date Deposited: 31 Jan 2018 19:32
Last Modified: 31 Jan 2018 19:32
URI: http://d-scholarship.pitt.edu/id/eprint/33287

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