Mechanisms of mRNA regulation by La-related protein 1

项目概要/摘要 细胞功能和体内平衡是通过 RNA 结合蛋白的精心编排来调节的，这些蛋白发挥着 在信使 RNA (mRNA) 转录、周转、亚细胞定位和最终翻译中发挥作用。我们 提议检查 RNA 结合蛋白 La 相关蛋白 1 (Larp1) 如何与非翻译蛋白相互作用 mRNA 的非编码区 (UTR)，将其分流至翻译抑制或激活的途径。许多 Larp1 与之相互作用的 RNA 编码对上皮间质转化 (EMT) 至关重要的蛋白质，EMT 是一种 引发侵袭性、转移性行为和耐药性的一系列变化。事实上，放松管制 Larp1 水平与卵巢癌、肺癌、肝癌和宫颈癌有关。因此，了解底层 Larp1 功能机制将有助于设计诊断工具和治疗目标。 Larp1 包含至少三个假定的 RNA 结合域，即 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端区域，称为DM15基序， 其序列仅出现在 Larp1 蛋白中。 Larp1 已被提议与 UTR 相互作用 mRNA 和多聚 A 结合蛋白 (PABP)，该蛋白调节 mRNA 的稳定性和翻译 mRNA。最近的数据表明 Larp1 将生长响应性 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 蛋白质和翻译相关因子；这些 mRNA 的 5' 末端含有寡嘧啶束 （5' 顶部）。我们假设 mRNA 5' 和 3' UTR 中特定 RNA 序列的结合通过 Larp1 调节其构象，从而调节其与聚 A 结合蛋白直接相互作用的能力。我们 将： 1) 确定 Larp1 的 DM15 基序和 5' TOP 之间相互作用的分子基础 多聚 A mRNA。 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 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来完成 利用现有晶型进行测定。目标 3 将采用跨学科方法 结合 Larp1-RNA 识别的体外生化表征和高通量测序 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机制，而且我们的结果还将展示新的模式 mRNA翻译调控。我们将为 Larp1 提供第一个结构数据。此外，我们还将揭晓 含有 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.