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Han, Kyoungha (2004) ALTERNATIVE SPLICING OF THE GRIN1 CI CASSETTE EXON: SILENCING MECHANISM. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Han, Kyounghakyhst2@pitt.eduKYHST2
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGrabowski, Paula Jpag4@pitt.eduPAG4
Committee MemberPeebles, Craig Lcpeebles@pitt.eduCPEEBLES
Committee MemberBrodsky, Jeffrey Ljbrodsky@pitt.eduJBRODSKY
Committee MemberWoolford,
Committee MemberArndt, Karenarndt@pitt.eduARNDT
Date: 23 September 2004
Date Type: Completion
Defense Date: 16 August 2004
Approval Date: 23 September 2004
Submission Date: 18 August 2004
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
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: alternative splicing
Other ID:, etd-08182004-103253
Date Deposited: 10 Nov 2011 20:00
Last Modified: 15 Nov 2016 13:49


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