Heterogenous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear and have been shown to be involved in multiple cellular processes. There are more than 20 members of hnRNP protein family and they all bind RNA. Some hnRNPs like the ones in hnRNP H family was shown to have sequence specific RNA binding activity. hnRNP H potein family includes hnRNP H1, H2. H3 and F. This study mapped the location of hnRNP H/F binding site on the SV40 Late (SVL) pre-mRNA relative to the polyA signal. The binding site is characterized down to five consecutive Guanines. Disruption of hnRNP H/F binding site on the SVL pre-mRNA had a negative effect on polyadenylation in vitro and this effect is translated to downregulated gene expression in Gfp based in vivo assays. The fact that this effect was seen with constructs having single polyA sites implicates a pronounced effect on alternative polyadenylation. The second part of this study involves microarray experiments with A20 B-cell lymphoma and AxJ Plasmacytoma cells. More than 400 genes were found to be differentially expressed in AxJ cells. While this group includes many known B-cell stage specific genes, there are also numerous novel differentially expressed transcripts. The list includes transcription, splicing and polyadenylation factors, too. Elongation factor RNA polymerase II 2 (ELL2) is induced in AxJ cells as assayed by microarray and RT-PCR experiments. The overexpression of hnRNP F was shown revert the B-cell clock, in so as far as secretory to membrane Immunoglobulin ratio is concerned. In an attempt to identify the genes underlying this mechanism, microarray experiments were preformed with hnRNP F overexpressing AxJ plasmacytoma cells and compared the gene expression profile with the one of mock transfected cells. There are about 30 differentially expressed transcripts upon forced expression of hnRNP F. Three B-cell stage specific genes were differentially expressed in hnRNP F overexpressing AxJ cells. ELL2 is among these genes. ELL2 is a known transcriptional elongation enhancer. A novel model regarding the secretory to membrane Immunoglobulin switch and involving ELL2 is proposed.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12082004-140239 |
| Date | 10 December 2004 |
| Creators | Alkan, Serkan |
| Contributors | Christine Milcarek, Neal DeLuca, Paula Grabowski, Baskaran Rajasekaran, Martin Schmidt |
| 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-12082004-140239/ |
| 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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