DEEP MUTATIONAL SCANNING IN VIVO OF AN RNA RECOGNITION MOTIF

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 RNA结合蛋白（RBP）介导RNA代谢的几乎所有方面，包括转录、加工、亚细胞定位以及翻译和衰变的调节。由于这些功能是多种细胞过程的基础，因此破坏RBP功能的遗传变化可能导致人类病理学，如神经系统疾病和癌症。RNA识别基序（RRM）是真核生物中最常见的RNA结合结构域。我们已经采取了一种方法，深度突变扫描，研究在体内功能的一个单一的RRM。该方法查询感兴趣域中每个位置的突变的相对适应度效应。在这种情况下，我们利用的必要性的功能聚（A）结合蛋白（Pab 1）的酵母活力。我们创建了一个酵母表达质粒库，在RRM 2结构域的Pab 1的平均三个突变，并转化到酵母表达野生型Pab 1的四环素抑制启动子下的质粒。在向培养物中加入四环素后，具有功能性Pab 1 RRM 2结构域的转化体存活，而非功能性突变体丢失。输入和选择的文库池中的RRM 2结构域准备好使用Illumina测序平台进行测序。我们预计，该实验的结果将有助于表征该结构域的RNA结合活性，以及尚未鉴定的对功能重要的蛋白质区域。此外，从这些实验中提取的数据可能用于预测与几种人类遗传疾病相关的RRM突变如何影响该结构域的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. RNA-binding proteins (RBPs) mediate virtually all aspects of RNA metabolism, including, transcription, processing, subcellular localization, and regulation of translation and decay. Since these functions underlie multiple cellular processes, genetic changes that disrupt RBP function can lead to human pathologies, such as neurological disorders and cancer. The RNA Recognition Motif (RRM) is the most common RNA-binding domain in eukaryotes. We have undertaken an approach, deep mutational scanning, to study the in vivo function of a single RRM. This approach queries the relative fitness effect of mutations in every position in the domain of interest. In this case we exploit the necessity of a functional Poly(A) binding protein (Pab1) for yeast viability. We created a library of yeast expression plasmids with an average of three mutations in the RRM2 domain of Pab1, and transformed the plasmids into yeast expressing a wild-type Pab1 under a tetracycline-repressible promoter. After addition of tetracycline to the culture, transformants that have a functional Pab1 RRM2 domain survive while nonfunctional mutants are lost. The RRM2 domain in the input and the selected library pools are ready to be sequenced using the Illumina sequencing platform. We anticipate that the results of this experiment will help to characterize the RNA-binding activity of this domain as well as yet unidentified protein regions that are important for function. Furthermore, the data extracted from these experiments might be used to predict how RRM mutations associated with several human genetic diseases affect the function of this domain.