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.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-09282005-152855 |
| Date | 20 October 2005 |
| Creators | Shell, Scott Allen |
| Contributors | Anuradha Ray, Ph.D., Sidney M. Morris, Jr., Ph.D., Martin C. Schmidt, Ph.D., Christine Milcarek, Ph.D., Scott Plevy, M.D. |
| 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-09282005-152855/ |
| 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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