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Mechanisms of ARE-mediated gene repression by Tristetraprolin and homologs

Abstract

The zinc finger protein Tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). While much attention has been directed towards understanding the decay process and the machinery involved, the translation repression role of TTP has remained poorly understood. My thesis research identified the cap-binding translation repression 4EHP-GYF2 complex as a co-factor of TTP. Immunoprecipitation and in vitro pulldown assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4ehp knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP target mRNAs as compared to wild-type MEFs. This work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a co-factor in translational repression and mRNA decay by TTP.

The human genome encodes for two TTP homologs, BRF1 and BRF2, both of which are capable of ARE binding and decay activation. I observed that TTP family proteins are differentially regulated during serum-activated G0-G1-S transition in NIH 3T3 cells. The G0-G1-S cell cycle transition is regulated at multiple levels to avoid erroneous or mistimed cell proliferation. Transition over the G0-G1-S cell cycle requires repression of Retinoblastoma (RB) proteins, which are central components of the CDK-RB-E2F checkpoint pathway controlling in this transition. I identified ARE-containing Rb, Rbl1 and Rbl2 mRNAs encoding retinoblastoma proteins as targets of degradation by TTP family proteins. Depletion of TTP family proteins results in reduced expression of E2F transcription targets, and slower transition out of quiescence compared to control conditions. Together, this work demonstrates the involvement of mRNA decay in cell cycle progression.

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