A systems approach to understand translation and sRNA-mediated gene regulation in

描述（由申请人提供）：新月柄杆菌中的细胞分化作为细胞周期的函数发生，其中转录电路通过基因表达的不同阶段指导进展，以确保多个事件以时空调节的方式发生。尽管有令人信服的证据表明细胞周期电路对翻译的调节，但翻译在柄杆菌属细胞周期进程中的作用相对未被探索。该项目的目标是了解基因表达如何通过mRNA翻译来调节，以确保柄杆菌的细胞周期进程。为了理解这一过程，我将采用一种新的方法，核糖体分析，通过测量核糖体保护的mRNA片段的数量以及高通量测序的核糖体位置来监测全基因组翻译的变化。此外，我将探讨一类新的基因，反式编码的小RNA，这是最近发现的表达在细胞周期的不同阶段的作用。这类基因通过RNA伴侣Hfq与mRNA碱基配对并正向或负向调节mRNA翻译和mRNA稳定性来发挥功能。我将使用核糖体分析与敲唐斯编码小RNA和Hfq的基因，以确定这些基因如何帮助翻译调控。此外，我将确定哪些sRNA及其mRNA靶点与调节因子Hfq物理相关，并通过使用交联免疫沉淀（HITS-CLIP）分离的RNA的高通量测序来确定RNA上的Hfq结合位点。该方法在体内UV交联Hfq核糖核蛋白复合物，然后使用核糖核酸酶保护来捕获结合的片段，并使用高通量测序来鉴定Hfq的sRNA和mRNA结合位点。通过这种广泛的方法，我将确定如何调节全局mRNA翻译，以确保基因表达的动态变化，从而驱动柄杆菌属的细胞周期进程。

DESCRIPTION (provided by applicant): Cell differentiation in Caulobacter crescentus occurs as a function of the cell cycle, where transcriptional circuitry guides progression through distinct phases of gene expression to ensure multiple events take place in a spatiotemporally regulated manner. Despite compelling evidence of the regulation of translation by the cell cycle circuitry, the role of translation is relatively unexplored in Caulobacter cell cycle progression. The goal of this project is to understand how gene expression is regulated by mRNA translation to ensure cell cycle progression in Caulobacter. In order to understand this process, I will employ a new method, ribosome profiling, to monitor genome wide changes in translation by measuring the number of ribosome-protected mRNA fragments as well as ribosome positions with high throughput sequencing. Additionally, I will explore the role of a new class of genes, trans encoded small RNAs, which were recently discovered to be expressed during distinct phases of the cell cycle. This class of genes functions by base pairing to mRNAs through the RNA chaperone Hfq and positively or negatively regulating mRNA translation and mRNA stability. I will use ribosome profiling with knock downs of genes encoding small RNAs and Hfq to identify how these genes aid in translation regulation. Additionally, I will determine which sRNAs and their mRNA targets physically associate with the regulator Hfq, and determine the Hfq binding sites on the RNAs by using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). This method UV crosslinks Hfq ribonucleoprotein complexes in vivo, then uses a ribonuclease protection to capture the bound fragments, and uses high-throughput sequencing to identify the sRNA and mRNA binding sites of Hfq. With this broad approach, I will determine how global mRNA translation is regulated to ensure the dynamic changes in gene expression that drive cell cycle progression in Caulobacter.