Quantifying native RNA dynamics during regulation

描述（由申请人提供）：基因调控是正常细胞功能、细胞分化和对环境刺激作出反应所必需的基本过程。此外，它在许多重要的病理学中起着重要作用，如癌症和病毒感染。转录后水平的调控在癌症中变得极其重要，与肿瘤发生的早期阶段和转移有关。在亚细胞水平上，它与mRNA定位和动力学、mRNA-蛋白质和mRNA-miRNA相互作用直接相关。目前，还没有在活的哺乳动物细胞中单个RNA或颗粒水平上研究天然mRNA的动态。其原因是缺乏在单个RNA或颗粒水平上对天然RNA进行成像的方法。为了解决这个问题，Santangelo实验室开发了一种单一的RNA敏感成像策略，最近发表在Nature Methods上。我们的方法包括两个部分：通过沃森-克里克配对与天然RNA结合的探针和通过链球菌溶血素O有效的胞质递送。我们的探针设计由四个高亲和力，耐核酸酶，线性核酸，标记有多个，高量子产率的荧光团连接在一起的链霉亲和素，通过生物素-链霉亲和素连接，其中链霉亲和素是探针核心。通过每个RNA结合多个探针实现的增强的信号背景比来鉴定靶RNA。以这种方式，以与GFP-RNA结合蛋白或肽系统类似的方式实现靶的鉴定，但是利用天然靶序列和显著更少的结合位点。在初步实验中，探针快速结合天然的胞质靶RNA（<10分钟），并且当与荧光融合蛋白结合使用时，允许首次证明单个RNA敏感性、RNA颗粒运输和动态RNA-蛋白质共定位研究。因此，本研究的短期目标是充分验证我们用于RNA运输研究的单分子敏感成像方法，并在单个RNA或颗粒水平上定量天然2-肌动蛋白mRNA的动态，同时运输到翻译位点，这是一个已知的调控过程，与细胞运动高度相关。我们还将这些结果与来自质粒的RNA的结果进行比较。我们的长期目标是双重的，1）开发使用mRNA动力学测量来间接测量mRNA功能的能力，以及2）利用这种成像方法与siRNA、荧光抗体染色和融合蛋白结合，以确定反式作用因子（如RNA结合蛋白和miRNA）的作用，在一系列调控过程中，在转录后水平上调控基因表达。这将使新的方法能够干预和控制这些过程，特别是用于预防癌细胞转移或使这些细胞更容易接受治疗。 公共卫生相关性：基因调控在许多重要的生物学问题和过程中起着关键作用，例如癌症发病机制和干细胞分化，病毒感染和神经回路的组装。最近，RNA动力学已被强烈地链接到转录后基因调控通过本地翻译的机制，并通过多聚核糖体，P-体，和应力颗粒之间的mRNA的动态运输。在这项授权中，我们将充分验证一个单分子敏感的，荧光的，天然RNA成像方法，量化天然RNA的动力学，同时贩运到当地的翻译位点，并将这些结果与以前的质粒衍生的RNA动力学进行比较，以确认使用质粒衍生的RNA作为天然RNA动力学的模型或识别运动的差异，可能是由于顺式作用序列的变化，RNA结构或通过不同的生物合成途径旅行的结果。

DESCRIPTION (provided by applicant): Gene regulation is a fundamental process necessary for normal cell function, cell differentiation, and for responding to environmental stimuli. In addition, it plays a significant role in many important pathologies, such as cancer and viral infections. Regulation at the post-transcriptional level has become extremely important in cancer, linked with the early stages of tumorigenesis and with metastasis. On the subcellular level, it has been directly linked to mRNA localization and dynamics, mRNA-protein and mRNA-miRNA interactions. Currently, there have been no studies of native mRNA dynamics on the single RNA or granule level in living mammalian cells. The reason for this has been the lack of methods to image native RNAs on the single RNA or granule level. To address this issue, the Santangelo lab has developed a single RNA sensitive imaging strategy recently published in Nature Methods. Our methodology consists of two parts; the probes, which bind to native RNA via Watson-Crick pairing, and efficient cytosolic delivery via streptolysin O. Our probe design consists of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage, where streptavidin is the probe core. Target RNA is identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA. In this way, the identification of target is achieved in an analogous way to that of GFP-RNA binding protein or peptide systems, but native target sequences are utilized and significantly fewer binding sites. In preliminary experiments, probes bound rapidly to native, cytosolic target RNA (<10 minutes), and allowed for the first demonstrations of single RNA sensitivity, RNA granule trafficking, and dynamic RNA- protein colocalization studies, when used in conjunction with fluorescent fusion proteins. Therefore, the short term goal of this study is to fully validate our single molecule-sensitive imaging approach for the study of RNA trafficking, and quantify the dynamics of native 2-actin mRNA on the single RNA or granule level while trafficking to translation sites, a known regulatory process, highly associated with cell motility. We will also compare these results with those from plasmid-derived RNAs. Our long term goals are two-fold, 1) to develop the ability to use mRNA dynamics measurements to indirectly measure mRNA function, and 2) to utilize this imaging methodology in conjunction with siRNA, fluorescent antibody staining and fusion proteins, to determine the roles of trans-acting factors, such as RNA binding proteins and miRNAs, in the regulation of gene expression on the post-transcriptional level during a range of regulatory processes. This should enable new methods to intervene and control these processes, especially as applied to preventing cancer cell metastasis or making these cells more susceptible to treatment. PUBLIC HEALTH RELEVANCE: Gene regulation plays a critical role in many important biological problems and processes, such as cancer pathogenesis and stem cell differentiation, viral infections, and the assembly of neural circuits. Recently, RNA dynamics have been strongly linked to post-transcriptional gene regulation through the mechanisms of local translation and through the dynamic trafficking of mRNA between polysomes, P-bodies, and stress granules. In this grant we will fully validate a single molecule sensitive, fluorescent, native RNA imaging methodology, quantify the dynamics of native RNAs while trafficking to local translation sites, and compare these results with previous plasmid-derived RNA dynamics, in order to either confirm the use of plasmid-derived RNA as a model of native RNA dynamics or identify differences in motion, possibly due to variations in cis-acting sequences, RNA structure or as a consequence of traveling through a different biogenesis pathway.