Regulation and function of the RNA-binding protein CELF1
2012-07
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Regulation and function of the RNA-binding protein CELF1
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2012-07
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Post-transcriptional gene regulation is a crucial aspect of the cellular control mechanisms that ensure the accurate expression of genetic information. Coordinate regulation of the rate of decay of networks of functionally related mRNA provides a cell the ability to quickly and precisely alter its transcriptome in response to environmental signals. An important regulator of mRNA decay is the RNA binding protein CUGBP1 and Etr3 Like Factor 1 (CELF1). CELF1 binds to a GU-rich element (GRE) harbored in target transcripts and promotes their rapid decay through the recruitment of RNA decay enzymes. The work presented in this thesis focused on elucidating the function and regulation of CELF1 mediated mRNA decay through the following objectives:
The first objective was to elucidate the network of transcripts that are regulated by CELF1. We utilized RNA-immunoprecipitation targeting CELF1 followed by microarray analysis of the co-precipitated transcripts to determine that CELF1 targets in HeLa cells and primary human T cells. We found that CELF1 targeted a network of approximately 600 transcripts in HeLa cells, 1300 transcripts in resting T cells, and 150 transcripts in stimulated T cells. Functional analysis of these transcripts revealed that CELF1 transcripts code for proteins involved in cellular proliferation, apoptosis, actin-based motility, and post-transcriptional regulation. Bioinformatic analysis of the CELF1 target transcripts revealed enrichment of the previously defined GRE, as well as a GU-repeat motif, in the 3'UTR of transcripts. We further showed that this GU-repeat motif conferred CELF1-mediated rapid decay to otherwise stable reporter transcripts, and thus re-defined the GRE as a UGU[G/U]UGU[G/U]UGU sequence occurring in the context of the 3'UTR. Through investigation of the decrease in the CELF1 target transcript population, we found that CELF1 underwent an activation dependent phosphorylation event. We found that this phosphorylation event inhibited CELF1 mRNA binding, and correlated with the increased half-life and abundance of CELF1 resting cell targets during T cell activation.
The second objective of this thesis was to investigate whether aberrant regulation of CELF1 mediated mRNA decay may play a role in viral oncogenesis. To investigate this, we utilized a model of KSHV infection whereby HeLa cells were transfected with the oncogenic KSHV encoded GPCR. We found that the vGPCR caused CELF1 phosphorylation, likely in a MEK/ERK dependent fashion. We showed that this phosphorylation correlated with reversal of CELF1 mediated mRNA decay, while maintaining CELF1s ability to bind to the GRE. Investigation into the mechanism of CELF1 inhibition by the vGPCR revealed that phosphorylation of CELF1 through vGPCR signaling may inhibit CELF1s recruitment of the deadenylase PARN, thus inhibiting CELF1 mRNA decay. The inhibition of CELF1 mediated decay is expected to result in the stabilization of a network of proliferation and apoptosis regulatory transcripts, leading to dysregulation of these pathways and potentially contributing to an oncogenic phenotype.
Finally, we studied the affect of alternative polyadenylation (APA) during the early timepoints of T cell stimulation on mRNA decay networks. We utilized high-throughput sequencing technologies to globally quantify the APA landscape at zero, six, and 24 hours of in vitro T cell stimulation. Using this data we were able to confirm previous reports that T cell stimulation promotes preferential usage of proximal polyA sites, but our results suggest that this occurs much earlier than previously reported. Additionally, we present data suggesting that GREs and AU-rich elements (AREs) are much more likely to be excised than included in a 3'UTR as a result of APA, suggesting preferential regulation of these mRNA decay networks. Finally, we present data suggesting that the temporal pattern of GRE and ARE regulation by APA is different, suggesting functional independence in the ARE and GRE containing networks of transcripts.
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University of Minnesota Ph.D. dissertation. July 2012. Major: Microbiology, Immunology and Cancer Biology. Advisor: Paul Bohjanen, MD PhD. 1 computer file (PDF); xii, 196 pages.
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Beisang, Daniel Joseph. (2012). Regulation and function of the RNA-binding protein CELF1. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/133170.
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