Uncovering the role of individual RNA helicases in cancer through specific chemical inhibition

 描述(由申请人提供)：有效的翻译调控是人类细胞的一个标志，因为它提供了对细胞生长和增殖速度的精确控制。癌细胞通常表现出翻译控制的失调，通常是通过核糖体蛋白和rRNAs的表达增加，或者通过促进生长和增殖的途径增加信号。其中一个信号级联途径，PI3K-mTOR途径，部分通过激活起始因子eIF4A来调节蛋白质的合成，eIF4A是一种RNA解旋酶，通过信使RNA5‘-非翻译区(5’-UTR)内的二级结构的局部解旋来增加翻译起始的速率，并允许它们在核糖体上翻译。最近的研究已经发现了多种在mRNA翻译中发挥非冗余作用的其他RNA解旋酶，但这些酶的底物特异性及其在肿瘤发生中的作用尚不清楚。这项建议旨在开发新的化学工具来研究RNA解旋酶，并将使用这些工具来揭示RNA解旋酶调节翻译的范围。我们假设具有共同功能的基因在其mRNA的5‘-UTR区含有保守的二级结构元件，并且这种结构的RNA被特定的RNA解旋酶识别，这反过来对调节该基因亚集的翻译是至关重要的。目标1将开发一种基于共价互补的“电泳性敏感”化学遗传方法，以在具有小分子的细胞环境中特异性地抑制单个RNA解旋酶。初步研究表明，半胱氨酸可以突变为RNA解旋酶的ATP结合部位，并且这种电泳性(ES)RNA解旋酶对基于NMS-859的小分子亲电体是唯一敏感的。我们将首先测试这种半胱氨酸突变在所有RNA解旋酶亚群中的普适性，以确保它们在生化和细胞中都是活跃的。同时，我们将合成NMS-859的衍生物，以提高ES RNA解旋酶的效力，同时减少脱靶反应。目标2将评估单个RNA解旋酶在翻译控制中的作用。与翻译控制有关的RNA解旋酶的电敏感版本(eIF4A、DDX3X、DDX4、DDX6、DHX29、DDX43)将通过CRISPR介导的基因组工程单独导入其他等基因的细胞系。在ES RNA解旋酶抑制剂存在的情况下，核糖体图谱将被用来揭示每个单独的RNA解旋酶对翻译的调节范围。这一建议的完成将阐明mRNA二级结构和RNA解旋酶对翻译的调控，并提供一系列新的治疗靶点来限制癌症中翻译的失调。

 DESCRIPTION (provided by applicant): Efficient regulation of translation is a hallmark of human cells as it affords precise control over rates of cellular growth and proliferation. Cancer cells commonly display a dysregulation of translational control, often through increased expression of ribosomal proteins and rRNAs or increased signaling through pro-growth and proliferation pathways. One of these signaling cascades, the PI3K-mTOR pathway, regulates protein synthesis in part through its activation of the initiation factor eIF4A, an RNA helicase which increases rates of translation initiation through the local unwinding of secondary structure within the 5'-untranslated region (5'-UTR) of messenger RNAs and allows for their translation at the ribosome. Recent studies have identified multiple other RNA helicases that play non-redundant roles in mRNA translation, but the substrate specificity of these enzymes, as well as their roles in tumorigenesis, is not well understood. This proposal aims to develop novel chemical tools for the study of RNA helicases and will use these tools to uncover the scope of regulation of translation by RNA helicases. We hypothesize that genes with common functions contain conserved secondary structural elements within the 5'-UTR of their mRNA and that this structured RNA is recognized by a specific RNA helicase, which in turn is critical for regulating the translation of this gene subset. Aim 1 will develop an "electrophile-sensitive" chemical genetic approach based on covalent complementarity to specifically inhibit a single RNA helicase in a cellular environment with a small molecule. Preliminary studies suggest that a cysteine can be mutated into the ATP-binding site of RNA helicases and that this electrophile- sensitive (ES) RNA helicase is uniquely sensitive to NMS-859-based small molecule electrophiles. We will first test the generalizability of this cysteine-mutation across all subsetsof RNA helicases to ensure that they are active biochemically and in cells. Concurrently, we will synthesize derivatives of NMS-859 to increase potency for ES RNA helicases while reducing off-target reactivity. Aim 2 will assess the role of individual RNA helicases in translational control. Electrophile-sensitive versions of the RNA helicases implicated in translational control (eIF4A, DDX3X, DDX4, DDX6, DHX29, DDX43) will be individually introduced into otherwise isogenic cell lines with CRISPR-mediated genome engineering. Ribosomal profiling in the presence of ES RNA helicase inhibitors will be used to uncover the scope of regulation of translation by each individual RNA helicase. Completion of this proposal will elucidate the regulation of translation by mRNA secondary structure and RNA helicases, and provide a set of novel therapeutic targets to limit the dysregulation of translation in cancer.