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

信使核糖核酸的稳定性在不同的信使核糖核酸物种之间有很大的不同，并且在决定基因表达水平方面起着重要作用。不同的信使核糖核酸衰减率是由信使核糖核酸分子中特定的顺式作用元件决定的。富含AU的元件(ARE)是哺乳动物细胞中最常见的顺式元件，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本的3UTRs中找到。现在已经清楚的是，ARES可能是大多数不稳定的mRNAs降解的原因，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。许多蛋白质已被描述为结合ARES(ARE结合蛋白(ARE-BP)。有些是促进信使核糖核酸衰变的因子，有些则是稳定因子。此外，这些蛋白质中的一些在破坏或稳定mRNAs方面的功能依赖于细胞环境和/或表达的蛋白质异构体。 我们首先重点鉴定了三个ARE-BPS、ELAVL1/HUR、ZFP36/TTP和HNRNPD/AUF1的目标mRNA结合位点，已知它们参与了短半衰期信息的清除。我们发现，ZFP36结合和负调控的靶标包括编码免疫功能和癌症所需蛋白质的转录本，以及编码其他限制性商业惯例的转录本。与野生型小鼠细胞相比，在ZFP36基因敲除的小鼠细胞中，mRNA半衰期增加的基因对我们的人类ZFP36靶点有显著的富集性。我们在1,313个基因中鉴定了数千个重叠的ZFP36和ELAVL1结合位点，并发现ZFP36通过特定的AU富含序列降解转录本，代表ELAVL1与富含U的序列相互作用以稳定转录本的子集。在一项分析HNRNPD调控作用的相关研究中，我们发现，正如基于对单个靶标转录稳定性的研究所预期的那样，HNRNPD降低了许多靶标RNA的稳态水平。然而，令人惊讶的是，HNRNPD出人意料地提高了几个编码DNA维持蛋白的目标mRNAs的稳态水平。因此，HNRNPD保持了基因组的完整性，这与AUF1诱导的防止细胞过早衰老是一致的。 目前，我们正在关注KH型剪接调节蛋白(KHSRP)，这是一种与发育过程和miRNA生物发生有关的ARE-BP，以及它在破坏目标转录本稳定方面的作用。我们将把PAR-CLIP和RNAseq与基于CRISPR-Cas的基因工程相结合，以获得KHSRPs在转录后基因调控中广泛而重要的作用的完整概述。

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 ELAVL1 interacts with 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-elicited prevention of premature cellular senescence. Currently, we are focusing on the KH-type splicing regulatory protein (KHSRP), an ARE-BP which is 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 KHSRPs broad and vital role in post-transcriptional gene regulation.