In vertebrates, alternative splicing regulates gene expression in a cell- and developmental stage-specific manner, producing functional diversity of the corresponding proteins. Although its importance has been underscored, control mechanisms of alternative splicing are poorly understood. The N-methyl-D-aspartate receptor NR1 subunit (NMDAR1, GRIN1) contains three cassette exons (NI, CI and CII), and which are specifically expressed in mammalian brain. In this study, a minigene splicing reporter system of the CI cassette exon was utilized to study its splicing silencing mechanism in mammalian cell lines. This work focuses primarily on the identification of exonic UAGG motifs and a 5 splice site region G cluster. Individual motifs play a silencing role but the combination of all three is required for strong silencing. Whereas insertion of an extra UAGG motif in the exon shows almost complete silencing, removal of all three silencers results in loss of silencing. Therefore, the UAGG and G cluster motifs confer flexibility of control of CI cassette exon splicing and the number of the motifs determines the silencing strength in various tissues. The UAGG motif interacts with hnRNP A1 and the strong silencing of hnRNP A1 requires the G cluster. The splicing enhancing role of hnRNP F or H/H is identified, and this effect largely requires the G cluster. Using bioinformatics searches, new groups of skipped exons containing the UAGG and GGGG (as a G cluster) motifs are identified from the human and mouse genomes. This study suggests that hnRNP H1 and H3 (HNRPH1 and HNRPH3) may be auto-regulated at the level of splicing. Overall, this work provides evidence for a splicing silencing mechanism that is important for the tissue-specificity of the CI cassette exon. This work also shows that the motif pattern can be used computationally to identify additional skipped exons that contain combinations of UAGG and GGGG motifs.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08182004-103253 |
| Date | 23 September 2004 |
| Creators | Han, Kyoungha |
| Contributors | Paula J Grabowski, Craig L. Peebles, Jeffrey L Brodsky, Karen Arndt, John Woolford |
| 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-08182004-103253/ |
| 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. |
Page generated in 0.0184 seconds