Quantifying native RNA dynamics during regulation

描述(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过程中发挥着重要作用，如癌症和病毒感染。转录后水平的调控在癌症中已变得极其重要，与肿瘤形成的早期阶段和转移有关。在亚细胞水平上，它与mRNA的定位和动态、mRNA-蛋白质和mRNA-miRNA的相互作用直接相关。目前，还没有关于活的哺乳动物细胞中单个RNA或颗粒水平上的天然mRNA动力学的研究。其原因是缺乏在单个RNA或颗粒水平上成像本地RNA的方法。为了解决这个问题，桑坦格罗实验室开发了一种单一的RNA敏感成像策略，最近发表在《自然方法》杂志上。我们的方法由两部分组成：通过Watson-Crick配对与天然RNA结合的探针和通过链霉亲和素O高效的胞浆递送。我们的探针设计包括四个高亲和力、抗核酸酶的线性核酸，用多个高量子产率的荧光团标记，通过生物素-链亲和素连接在一起，其中链霉亲和素是探针的核心。靶RNA是通过每个RNA结合多个探针而获得的增强的信号与背景比来识别的。通过这种方式，靶标的鉴定以类似于GFP-RNA结合蛋白或肽系统的方式实现，但使用了天然的靶标序列，并且结合位点显著减少。在初步实验中，探针迅速结合到天然的胞浆目标RNA(10分钟)，并允许首次展示单个RNA的敏感性、RNA颗粒运输和动态RNA-蛋白质共定位研究，当与荧光融合蛋白一起使用时。因此，这项研究的短期目标是充分验证我们的单分子敏感成像方法用于RNA运输的研究，并在单个RNA或颗粒水平上量化天然2-肌动蛋白mRNA在运输到翻译位点时的动态，这是一个已知的调控过程，与细胞运动高度相关。我们还将把这些结果与来自质粒衍生的RNA的结果进行比较。我们的长期目标有两个，1)发展利用mRNA动力学测量间接测量mRNA功能的能力，2)结合siRNA、荧光抗体染色和融合蛋白，利用这种成像方法，确定反式作用因子，如RNA结合蛋白和miRNAs，在一系列调控过程中，在转录后水平上调节基因表达的作用。这将使新的方法能够干预和控制这些过程，特别是在应用于预防癌细胞转移或使这些细胞更容易接受治疗时。 与公共卫生相关：基因调控在许多重要的生物学问题和过程中发挥着关键作用，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，通过局部翻译机制以及通过多聚体、P小体和应激颗粒之间的mRNA动态转移，RNA动态与转录后基因调控密切相关。在这项资助中，我们将全面验证一种单分子敏感的、荧光的天然RNA成像方法，量化天然RNA运输到本地翻译位点时的动力学，并将这些结果与以前的质粒源RNA动力学进行比较，以便确认使用质粒源RNA作为天然RNA动力学的模型，或识别运动的差异，这可能是由于顺式作用序列、RNA结构的变化或通过不同的生物发生途径造成的。

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.