The role of AU-rich element binding proteins in shaping target mRNA expression

mRNA的稳定性从一个mRNA物种到另一个物种变化很大，并且在决定基因表达水平方面起着重要作用。差异mRNA衰减率由mRNA分子内的特定顺式作用元件决定。富含AU的元件（ARE）是负责哺乳动物细胞中快速mRNA衰变的最常见的顺式元件，并且可以在编码细胞因子、趋化因子、转录因子、原癌基因和细胞周期调节因子的短寿命转录物的3UTR中发现。现在清楚的是，战神可以解释大多数不稳定mRNA的降解，并且mRNA半衰期的调节在基因表达的控制中起着至关重要的作用。已经描述了许多蛋白质结合战神（ARE结合蛋白（ARE-BP））。有些是mRNA衰变促进因子，而另一些是稳定因子。此外，这些蛋白质中的一些在去稳定或稳定mRNA中的功能取决于细胞环境和/或表达的蛋白质同种型。 我们首先专注于鉴定已知参与清除短半衰期信息的三种ARE-BP（ELAVL 1/HuR、ZFP 36/TTP和HNRNPD/AUF 1）的靶mRNA结合位点。我们发现，ZFP 36结合和负调控的靶点包括编码免疫功能和癌症所必需的蛋白质的转录本，以及编码其他RBP的转录本。与野生型小鼠细胞相比，在ZFP 36敲除小鼠细胞中具有增加的mRNA半衰期的基因对于我们的人ZFP 36靶标显著富集。我们在1，313个基因中鉴定了数千个重叠的ZFP 36和ELAVL 1结合位点，并发现ZFP 36通过特定的富含AU的序列降解转录物，这代表了ELAVL 1与其相互作用以稳定转录物的富含U的序列的子集。在一项分析HNRNPD调节作用的相关研究中，我们发现，正如基于对单个靶转录物稳定性的研究所预期的那样，HNRNPD降低了许多靶RNA的稳态水平。然而，令人惊讶的是，HNRNPD出乎意料地增强了几种编码DNA维持蛋白的靶mRNA的稳态水平。因此，HNRNPD保留了基因组的完整性，这与导致细胞过早衰老的AUF 1损失一致。 目前，我们正专注于KH型剪接调节蛋白（KHSRP），一个ARE-BP涉及发育过程和miRNA的生物合成，除了其提出的作用，在不稳定的目标转录。我们将PAR-CLIP和RNAseq与基于CRISPR-Cas的基因工程相结合，以全面了解KHSRP在转录后基因调控中发挥的广泛而重要的作用。

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.