Shell, Scott Allen
(2005)
REGULATION OF THE 64-kDA SUBUNIT OF CLEAVAGE STIMULATORY FACTOR ACTIVITY IN MACROPHAGE AND B LYMPHOCYTE mRNA 3'-END PROCESSING.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Eukaryotic pre-mRNA is processed within the 3'-untranslated region (3'-UTR) resulting in cleavage and polyadenylation. The efficiency of the cleavage reaction is dependent on the binding activity of the 64-kDa subunit of CstF, CstF-64, to the pre-mRNA and is increased with elevated levels of CstF-64. There is evidence that alternative polyadenylation occurs in the presence of increased CstF-64. Our results showed that CstF-64 levels increased with LPS stimulation of RAW 264.7 macrophages while the expression of other pre-mRNA processing factors remained unchanged. Because of the evidence that several macrophage genes exhibit alternative polyadenylation and post-transcriptional regulation under LPS stimulation, we used a reporter mini-gene to identify alternative polyadenylation in LPS-stimulated RAW macrophages. Upon LPS stimulation we measured a 2.5-fold increase in proximal poly(A) site selection that correlated with elevated levels of CstF-64. Forced expression of CstF-64 demonstrated similar alternative polyadenylation. Microarray analysis demonstrated 515 genes changed expression with LPS stimulation, 15 of which also changed with CstF-64 over-expression. A closer analysis of 5 of these 15 genes demonstrated alternative polyadenylation within their 3'-UTR. Closer analysis of the 3'-UTRs showed putative AU-rich regulatory elements. There is also evidence that pre-mRNA processing is coupled with transcription. Previous work has shown that the carboxy-terminal domain (CTD) of RNAP-II is necessary for 3'-end processing, that CstF binds to RNAP-II CTD and that this binding is CTD phosphorylation dependent. Because our lab has also shown that increases in CstF-64 binding activity upon B-cell differentiation causes alternative polyadenylation on the Ig heavy chain gene and occurs in the absence of CstF-64 increases, we believe that the local concentration of CstF-64 to the nascent pre-mRNA is increased in plasma cells through the phosphorylation-dependent recruitment of CstF-64 to RNAP-II CTD. Using chromatin immunoprecipitation (ChIP), we measured an increase in Serine-2 and Serine-5 phosphorylation of the RNAP-II CTD at the promoter and variable regions of the Ig heavy chain gene in plasma cells compared to memory B cells. We believe that this increase in RNAP-II CTD phosphorylation plays a role in either increased transcription of the Ig heavy chain gene or recruitment of pre-mRNA processing factors to the transcriptional machinery.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
20 October 2005 |
| Date Type: |
Completion |
| Defense Date: |
14 September 2005 |
| Approval Date: |
20 October 2005 |
| Submission Date: |
28 September 2005 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Immunology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Alternative polyadenylation; Cleavage Stimulatory Factor; macrophage; post-transcriptional gene regulation; pre-mRNA 3'-end processing |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-09282005-152855/, etd-09282005-152855 |
| Date Deposited: |
10 Nov 2011 20:02 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9400 |
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