Using Fitness Landscapes to assess RNA Structure-Function Relationships From Inside the Cell

摘要 RNA是一个重要的治疗靶点，然而大多数RNA序列结构功能分析是在 尽管两种环境之间有实质性的差异，但在体外，而不是在细胞的背景下。这个 这项建议的目的是利用局部适应度来探索细胞内的RNA序列/功能关系 风景画。我们这项工作的模型系统是细菌RNA调节器，例如核糖开关，它是 抗菌靶标和研究RNA序列-功能关系的重要生物物理模型。 根据以前的文献研究和我们自己的初步数据，我们假设在体外 实验不能完美地捕捉到许多结构化生物功能所需的序列要求 RNA。为了解决体外测量的参数如何与细胞表达和生物体相对应 适合性，我们计划使用三个细菌RNA调节器作为模型系统：芽孢杆菌中的甘氨酸核糖开关 枯草杆菌，肺炎链球菌中的PyrR调节因子，以及肺炎链球菌中的FMN核糖开关。我们的 具体目标是：1)确定RNA调节器的哪些序列变化会影响生物体的适应性和 如何在不同的条件下改变这些条件，包括定义的培养介质、 抗生素和小鼠感染模型。2)建立细胞基因表达谱之间的关系 和生物表型。3)评估基因表达和生物体适合性如何与体外培养相对应 结构和功能分析。我们将通过为每个RNA创建局部适应环境来实现这些目标 调节器，其中所有单突变体和大多数双突变体被创建为一个池，并随后使用 高通量测序作为最终读数。将使用各种不同的方法评估各种变异体 技术包括：在枯草杆菌和肺炎链球菌存在的情况下进行有机培养比赛 靶向抗生素(肺炎链球菌)和小鼠感染模型(肺炎链球菌)；以及微流控 微集落的封装使基于FADS的细胞基因表达和体外评估成为可能 转录终止参数。在这些高吞吐量观察之后，将评估 一组突变体，以确认观察到的表型并检查体外和 在单元格内(SHAPE-MAP)。我们的方案利用了我们在以前的工作中发现的生物表型 是一种评估RNA序列-功能关系的创新方法，这种关系不能在任何 另一条路。所提出的工作的预期结果是第一个RNA调节序列之间的映射 以及包括生物表型、基因表达和体外终止在内的一系列变量 特点。这一贡献意义重大，因为它将使人们能够更好地解释 传统的体外和基因表达分析，为使用抗生素靶向RNA调节剂和 设计新颖的RNA调节剂。

Summary RNA is an important therapeutic target, yet most RNA sequence-structure-function analysis is conducted in vitro rather than in the context of the cell despite substantive differences between the two environments. The objective of this proposal is to explore RNA sequence/function relationships within the cell using local fitness landscapes. Our model systems for this work are bacterial RNA regulators, such as riboswitches, which are antimicrobial targets and important biophysical models for studying RNA sequence-function relationships. Based on previous studies in the literature and our own preliminary data, we hypothesize that in vitro experiments imperfectly capture the sequence requirements required for biological function for many structured RNAs. To address how in vitro measured parameters correspond with cellular expression and organismal fitness, we plan to use three bacterial RNA regulators as model systems: the glycine riboswitch in Bacillus subtilis, the pyrR regulator in Streptococcus pneumoniae, and an FMN riboswitch in S. pneumoniae. Our specific aims are: 1) Determine which sequence changes to an RNA regulator impact organismal fitness and how these may be altered under different conditions that include defined culture medium, the presence of antibiotics, and a mouse infection model. 2) Establish the relationship between cellular gene expression profile and organismal phenotype. 3) Assess how gene expression and organismal fitness correspond with in vitro structure and function assays. We will achieve these aims by creating local fitness landscapes for each RNA regulator wherein all single and most double mutants are created as a pool, and subsequently evaluated using high-throughput sequencing as a final readout. The pools of variants will be appraised using a variety of techniques including: organismal competitions in culture (B. subtilis and S. pneumoniae), in the presence of targeting antibiotics (S. pneumoniae), and in a mouse infection model (S. pneumoniae); as well as microfluidic encapsulation of microcolonies to enable FADS-based assessment of cellular gene expression and in vitro transcription termination parameters. These high-throughput observations will be followed by assessment of a subset of mutants to confirm the observed phenotypes and examine secondary structure both in vitro and within the cell (SHAPE-MaP). Our proposal leverages organismal phenotypes we discovered in previous work and is an innovative approach to assess RNA sequence-function relationships that cannot be assessed in any other way. The expected outcome of the proposed work is the first mapping between RNA regulator sequence and a range of variables including organismal phenotype, gene expression, and in vitro termination characteristics. This contribution is significant because it will enable better interpretation of results from traditional in vitro and gene expression assays to inform efforts to target RNA regulators with antibiotics and to design novel RNA regulators.