Mechanisms of mRNA regulation by La-related protein 1

项目摘要/摘要 细胞功能和动态平衡是通过精心设计的RNA结合蛋白来调节的，RNA结合蛋白扮演着 在信使RNA(信使RNA)转录、周转、亚细胞定位和最终翻译中的作用。我们 建议研究RNA结合蛋白La相关蛋白1(Larp1)如何与未翻译的 将其分流到翻译抑制或激活的途径。许多人 Larp1与之相互作用的RNA编码对上皮-间充质转化(EMT)至关重要的蛋白质，a 引发侵袭性、转移性行为和耐药性的一系列变化。事实上，放松对 Larp1水平与卵巢癌、肺癌、肝癌和宫颈癌有关。因此，理解潜在的 Larp1功能的机制将有助于设计诊断工具和治疗靶点。 Larp1至少包含三个假定的RNA结合域，即La-和RNA识别基序，这两个基序是 预测形成一个结构模块和一个高度保守的C-末端区域，称为DM15基序， 其序列只出现在Larp1蛋白中。Larp1已被提议与UTRs的 与多聚A结合蛋白(PABP)一起，调节细胞的稳定性和翻译 MRNAs。最近的数据表明，Larp1将生长反应mTOR激酶的信息传递到 通过与mTOR和编码核糖体的mRNAs直接相互作用来刺激核糖体的生物发生 蛋白质和翻译相关因子；这些mRNAs的5‘末端含有寡嘧啶小束 (最多5英尺)。我们假设，在mRNA的5‘和3’UTRs中，特定的RNA序列通过 Larp1调节其构象，因此其直接与聚A结合蛋白相互作用的能力。我们 将：1)确定Larp1的DM15基序与5‘TOP之间相互作用的分子基础 Poly-A基因的表达。2)确定Larp1的La-RRM模块对其RNA结合特异性的贡献。3) 确定Larp1的RNA结合域如何协同调节mRNA的结合和翻译。目标 1和2将通过体外生化和生物物理方法和结构完成 利用现有的晶型进行测定。目标3将采用跨学科的方法 结合Larp1-RNA识别和高通量测序的体外生化特征 分析细胞中与Larp1相互作用的RNA。 我们不仅将揭示直接的结构性机制，而且我们的结果还将展示新的 MRNA翻译调控。我们将为Larp1贡献第一个结构数据。此外，我们还将揭晓 包含DM15基序的Larp1特异性C-末端区域的功能。最后，我们将深入了解 Larp1翻译调控的基因本体论和RNA序列决定因素。

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.