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"Computational analyses of pre-mRNA polyadenylation and mRNA stability"

by
Ju Youn Lee
Interdisciplinary Biomedical Sciences Program

B.S. 1999, Sung Kyun Kwan University, Korea

M.S. 2004, New Jersey Institute of Technology




Thesis Advisor: Bin Tian, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology

Tuesday, July 6, 2010
MSB E-609b, 2:00 P.M.


Abstract

Pre-mRNA cleavage and polyadenylation is an essential step for the maturation of almost all eukaryotic mRNAs, and is tightly coupled with termination of transcription. Over half of all human and mouse protein-coding genes harbor multiple polyadenylation (polyA) sites that lead to mRNA variants containing different 3’ Untranslated Regions (3’UTRs) and/or encoding distinct protein sequences. The 3’UTRs of mRNAs contain cis-acting elements for post-transcriptional regulation of gene expression, such as mRNA localization, translation, and stability. This work involves analysis of data concerning evolution of alternative polyadenylation (APA), regulation of mRNA stability in mouse myoblast cells, regulation of APA in development, and impact of APA on cis elements in 3’UTRs.
First, we examined the conservation and divergence of different types of alternative polyA sites across human, mouse, rat, and chicken genomes. We found that the 3’-most polyA sites tend to be more conserved than upstream ones, whereas polyA sites located upstream of the 3’-most exon, termed intronic polyA sites, tend to be much less conserved. Genes with longer evolutionary histories are more likely to have APA, suggesting gain of polyA sites through evolution. We also found that non-conserved polyA sites are associated with transposable elements (TEs) to a much greater extent than conserved ones, albeit less frequently utilized. Different types of TEs have different propensities in their association with polyA sites. Some TEs encode polyA sites that are utilized by endogenous genes; some contribute to upstream or downstream cis elements for polyadenylation; and some contain sequences that have high tendency to give rise to polyA sites. This study established a conservation pattern for alternative polyA sites in several vertebrate species, and indicated that the 3’ end of genes can be dynamically modified by TEs through evolution.
Next, using microarrays, we established the decay rate for over 7,000 mRNAs expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3’UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. Using data from RNA immunoprecipitation with antibodies against CUGBP1 followed by microarray (RIP-Chip), we uncovered CUGBP1-associated mRNAs. These mRNAs also showed dramatic enrichment of GREs in their 3’UTRs and encode proteins linked with cell cycle, and intracellular transport. Taken together, this study systematically established cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrated that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.
Lastly, using EST and SAGE data, we found that mouse genes tend to express mRNAs with longer 3’UTRs as embryonic development progresses. This trend can also be detected in differentiation of C2C12 cells. We systematically analyzed cis elements encoded in constitutive and alternative 3’UTRs, and found that large fractions of miRNAs and mRNA stability elements are located in regions that can be regulated by APA.
Together, our results suggest that lengthening of 3’UTR can significantly augment post-transcriptional control of gene expression during embryonic development and cell differentiation, such as microRNA mediated regulation and mRNA stability.


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