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

描述(由申请人提供)：新月藻的细胞分化是细胞周期的函数，其中转录电路引导基因表达的不同阶段进行，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翻译是由细胞周期电路调节的，但翻译在Caulbacter细胞周期进程中的作用相对来说还没有被探索。这个项目的目标是了解基因表达是如何通过mRNA翻译来调节的，以确保Caulbacter的细胞周期进展。为了理解这个过程，我将使用一种新的方法，核糖体图谱，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基因组在翻译过程中的变化。此外，我将探索一类新基因的作用，反式编码的小RNA，最近被发现在细胞周期的不同阶段表达。这类基因的功能是通过RNA伴侣Hfq与mRNAs进行碱基配对，积极或消极地调节mRNA的翻译和mRNA的稳定性。我将使用核糖体图谱和敲除编码小RNA和HfQ的基因来确定这些基因是如何帮助翻译调节的。此外，我将确定哪些sRNAs及其mRNA靶标与调节因子Hfq物理上相关，并通过使用交联式免疫沉淀分离的RNA的高通量测序(HITS-CLIP)来确定RNAs上的Hfq结合位点。该方法在体内UV交联Hfq核糖核蛋白复合体，然后使用核糖核酸酶保护捕获结合的片段，并使用高通量测序来确定Hfq的sRNA和mRNA结合位点。通过这一广泛的方法，我将确定如何调控全球mRNA翻译，以确保驱动Caulbacter细胞周期进展的基因表达的动态变化。

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.