Mechanisms of mRNA regulation by La-related protein 1

项目概要/摘要 细胞的功能和稳态是由RNA结合蛋白的精心设计来调节的， 在信使RNA（mRNA）转录、周转、亚细胞定位和最终翻译中的作用。我们 我建议研究RNA结合蛋白La相关蛋白1（Larp 1）如何与未翻译的 mRNA的非编码区（UTR）将它们分流到翻译抑制或激活的途径中。许多 与Larp 1相互作用的RNA编码上皮-间质转化（EMT）关键蛋白， 一系列的变化，煽动入侵，转移行为和耐药性。事实上， Larp 1水平与卵巢癌、肺癌、肝癌和宫颈癌有关。因此，了解底层 Larp 1功能的机制将有助于设计诊断工具和治疗靶点。 Larp 1包含至少三个推定的RNA结合结构域，La-和RNA识别基序，它们是 预测形成一个结构模块，和一个高度保守的C-末端区域，命名为DM 15基序， 其序列只出现在Larp 1蛋白中。Larp 1已经被提出与以下的UTR相互作用： mRNA和多聚腺苷酸结合蛋白（PABP），该蛋白调节蛋白的稳定性和翻译， mRNA。最近的数据表明，Larp 1将来自生长反应性mTOR激酶的信息传递给 通过与mTOR和编码核糖体的mRNA直接相互作用刺激核糖体生物发生 这些mRNA的5'末端含有寡嘧啶片段 （5' TOPs）。我们假设，在mRNA的5'和3' UTR中的特异性RNA序列的结合是通过 Larp 1调节其构象，从而调节其与poly A结合蛋白直接相互作用的能力。我们 1）确定Larp 1的DM 15基序与5' TOP之间相互作用的分子基础， 多聚腺苷酸mRNA。2)确定Larp 1的La-RRM模块对其RNA结合特异性的贡献。第三章 确定Larp 1的RNA结合结构域如何合作调节mRNA结合和翻译。旨在 1和2将通过体外生物化学和生物物理方法和结构来实现 利用现有的晶体形式进行测定。目标3将采用跨学科方法 结合Larp 1-RNA识别的体外生物化学表征和高通量测序， 分析细胞中与Larp 1相互作用的RNA。 我们不仅将揭示直接的结构机制，而且我们的结果还将展示新的模式。 mRNA翻译调节。我们将为Larp 1提供第一个结构数据。此外，我们还将披露 含有DM 15基序的Larp 1特异性C末端区域的功能。最后，我们将深入了解 基因本体论和RNA序列决定因素的基础上的翻译调控Larp 1。

Project summary/Abstract Cellular function and homeostasis are regulated by careful choreography of RNA binding proteins, which play roles in messenger RNA (mRNA) transcription, turnover, subcellular localization, and ultimately translation. We propose to examine how the RNA-binding protein La-related protein 1 (Larp1) interacts with the untranslated regions (UTRs) of mRNAs to shunt them into pathways for translational repression or activation. Many of the RNAs with which Larp1 interacts encode proteins critical for the epithelial-mesenchymal transition (EMT), a series of changes that instigates invasive, metastatic behavior and drug resistance. Indeed, deregulation of Larp1 levels is linked to ovarian, lung, liver, and cervical cancer. Therefore, understanding the underlying mechanisms of Larp1 function will aid in designing diagnostic tools and treatment targets. Larp1 contains at least three putative RNA binding domains, the La- and RNA recognition motifs, which are predicted to form a structural module, and a highly conserved C-terminal region, named the DM15 motif, whose sequence only appears in Larp1 proteins. Larp1 has been proposed to interact with the UTRs of mRNAs and with poly-A binding protein (PABP), the protein that regulates the stability and translation of mRNAs. Recent data suggests that Larp1 relays information from the growth-responsive mTOR kinase to stimulate ribosome biogenesis through direct interactions with mTOR and with mRNAs encoding ribosomal proteins and translation-associated factors; the 5' terminal ends of these mRNAs contain oligopyrimidine tracts (5' TOPs). We hypothesize that the binding of specific RNA sequences in the 5' and 3' UTRs of mRNA by Larp1 modulates its conformation, and therefore its ability to directly interact with poly A-binding protein. We will: 1) Determine the molecular basis for the interaction between the DM15 motif of Larp1 and 5' TOP and poly-A mRNA. 2) Determine the contribution of the La-RRM module of Larp1 to its RNA binding specificity. 3) Determine how the RNA-binding domains of Larp1 cooperate to regulate mRNA binding and translation. Aims 1 and 2 will be accomplished through in vitro biochemical and biophysical approaches and structure determination taking advantage of existing crystal forms. Aim 3 will utilize an interdisciplinary approach combining in vitro biochemical characterization of Larp1-RNA recognition and high-throughput sequencing analysis of the RNAs that interact with Larp1 in cells. Not only will we reveal direct structural mechanisms, but our results will also demonstrate new modes of mRNA translation regulation. We will contribute the first structural data for Larp1. In addition, we will reveal the function of the Larp1-specific C-terminal region containing the DM15 motif. Finally, we will lend insight into the gene ontology and RNA sequence determinants underlying translational regulation by Larp1.