mRNA stability varies considerably from one mRNA species to another and plays an important role in determining levels of gene expression. Differential mRNA decay rates are determined by specific cis-acting elements within the mRNA molecule. The AU-rich element (ARE) is the most common cis element responsible for rapid mRNA decay in mammalian cells and can be found in the 3UTRs of short-lived transcripts encoding cytokines, chemokines, transcription factors, proto- oncogenes, and cell-cycle regulators. It is now clear that AREs may account for the degradation of most unstable mRNAs and that the regulation of mRNA half-life plays a crucial role in the control of gene expression. Numerous proteins have been described to bind AREs (ARE-binding protein (ARE-BP). Some are mRNA decay-promoting factors while others are stabilizing factors. In addition, the function of some of these proteins in destabilizing or stabilizing mRNAs is dependent on the cellular context and/or the expressed protein isoforms. We first focused on identifying the target mRNA binding sites of three ARE-BPs, ELAVL1/HuR, ZFP36/TTP, and HNRNPD/AUF1, known to be involved in clearance of short half-life messages. We found that targets bound and negatively regulated by ZFP36 included transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences with which ELAVL1 interacts to stabilize transcripts. In a related study dissecting the regulatory effects of HNRNPD we found, as anticipated based on studies on individual target transcript stability, that HNRNPD lowered the steady-state levels of numerous target RNAs. Surprisingly, however, HNRNPD unexpectedly enhanced the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Accordingly, HNRNPD preserved genomic integrity in agreement with the AUF1-loss leading to premature cellular senescence. Currently, we are focusing on the KH-type splicing regulatory protein (KHSRP), an ARE-BP implicated in developmental processes and miRNA biogenesis, in addition to its proposed role in destabilizing target transcripts. We will integrate PAR-CLIP and RNAseq with CRISPR-Cas based genetic engineering to gain a complete overview of the broad and vital role played by KHSRPs in post-transcriptional gene regulation.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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