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Prospective Identification of Translational Regulators in EMT

Joel R Neilson

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National Institutes of Health (NIH)
Post-transcriptional control of mRNA localization, stability, and translation is a fundamentally important, yet largely underexplored, mechanism of gene regulation that contributes to virtually every aspect of cellular physiology. In many cases, elements within the 3' untranslated region (UTR) of a given mRNA transcript confer responsiveness to microRNAs and/or binding proteins, which may dictate the fate of the transcript. One physiological process in which post-transcriptional regulation of gene expression has been demonstrated to play an important role is the epithelial to mesenchymal transition (EMT). EMT is thought to initiate tumor metastasis by altering the shape, invasiveness, and motility of carcinoma cells within the primary tumor. The breast epithelial cell line MCF10a undergoes EMT in response to treatment with the cytokine TGF-ß. Using density gradient fractionation and Next Generation Sequencing, we have examined polysomal enrichment and depletion in MCF10a cells in both the epithelial and mesenchymal states. Within our model system, we have identified 54 gene products that are preferentially translated (4-fold or more enrichment over total mRNA) in the mesenchymal state. Among these gene products is SNAI1, a known driver of EMT in several experimental systems. The nature of the mechanism driving this polysomal enrichment, and whether the remainder of the observed gene products contribute to the initiation or maintenance of the mesenchymal state, are both unclear. The proposed research is designed to assess the feasibility of a template for prospective identification of nove translational regulators within our system. Our hypothesis is two-fold: firstly, that these gene products functionally contribute to the initiation or maintenance of the mesenchymal state, and secondly, that their preferential translation is modulated by the interaction of a common factor with the 3' UTRs of these transcripts. To test these hypotheses, we will employ paired functional genomic approaches that are mutually informative but non-interdependent. Via overexpression and RNAi knockdown, we will determine whether the identified gene products are necessary and sufficient for EMT in three distinct models of this process. In parallel, we will employ a novel, scalable, transposon-based reporter platform to define for which of these mRNA transcripts the 3' UTR confers the translational control. Whether a cis-regulatory element common to many of these transcripts underlies this control will also be directly assessed. Finally, we will use biochemical and molecular methods to prospectively identify and characterize putative trans-regulators binding these putative cis-elements, under the rationale that these trans-regulators may integrate post-transcriptional gene regulation in the EMT programs. Novel regulators of EMT identified in our model system, both direct effectors and the trans-factors that regulate them, are potential targets for therapeutic intervention aimed at preventing metastasis of early-stage cancers. Further, successful implementation of our approach will result in a generalized template that can be leveraged in other cell-based models of tumorigenesis.

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