Identifying cis and trans factors required for microRNA function

描述(申请人提供)：基因表达调控是生物学的基础，基因表达的变化是人类疾病的常见原因。基因调控通常是在转录水平上研究的，但人们越来越认识到转录后对基因表达的调节。在包括动物在内的许多真核生物中，microRNAs(MiRNAs)指导着大部分转录后调控。MicroRNAs是一种短的、非编码的调控RNA，它通过碱基对靶信使RNAs(MRNAs)转录后抑制基因的表达。在人类中，miRNAs参与了广泛的生物途径，此外，干扰miRNAs的突变或其靶向，与越来越多的人类疾病有关，包括各种形式的癌症。自从miRNAs最初被发现以来，人们已经对miRNAs的生物发生、调控和作用模式有了很多了解，但我们的知识是不完整的，识别miRNA生物学中涉及的新因素将有助于我们更好地了解这些重要的调控分子在人类中的功能。我们的第一个目标是识别涉及miRNA生物学的新的蛋白质因子；我们的方法使用RNAi来抑制每个人类基因，并使用一种新的基于细胞的筛选策略来识别其抑制改变miRNA功能的基因。在我们的第二个目标中，我们专注于提高我们识别每个miRNA的目标mRNAs的能力，这仍然是miRNA生物学中的一个基本问题，既是为了更好地了解miRNAs作为一个类别的机制，也是为了了解单个miRNAs的生物学功能。尽管miRNA靶标预测的计算方法越来越复杂，但持续的进展受到验证和改进模型的合适实验技术和数据的可用性的限制。由于最先进的预测包含许多错误，但非常有用和广泛使用，我们有动力设计一个改进的靶标识别实验框架；我们的方法允许在最小扰动的内源细胞环境中高通量评估miRNA靶点。人们越来越广泛地认识到miRNAs对生物学许多领域的影响，这表明这种努力可能具有广泛的适用性。

DESCRIPTION (provided by applicant): Regulation of gene expression is fundamental to biology, and alterations in gene expression are a frequent cause of human disease. Gene regulation is typically investigated at the level of transcription, yet there is a growing recogniton of consequential post-transcriptional modulation of gene expression. In many eukaryotes, including animals, microRNAs (miRNAs) direct much of post-transcriptional regulation. MicroRNAs are short, non-coding, regulatory RNAs that post-transcriptionally repress gene expression by basepairing to target messenger RNAs (mRNAs). In humans, miRNAs contribute to a wide variety of biological pathways, moreover, mutations perturbing miRNAs, or their targeting, are implicated in a growing number of human diseases, including a variety of forms of cancer. Since their initial discovery, much has been learned about the biogenesis, regulation and mode of action of miRNAs, however, our knowledge is incomplete and the identification of novel factors involved in miRNA biology will help us better understand how these important regulatory molecules function in humans. Our first aim is to identify new protein factors involved in miRNA biology; our approach uses RNAi to inhibit each human gene and a novel cell-based screening strategy to identify genes whose inhibition alters miRNA function. In our second aim, we focus on improving our ability to identify the target mRNAs for each miRNA, this remains a fundamental question in miRNA biology, both to better understand the mechanisms of miRNAs as a class, and to understand the biological functions of individual miRNAs. Despite the increasing sophistication in computational approaches to miRNA target prediction, continued progress is limited by the availability of suitable experimental techniques and data to validate and refine models. Because state-of-the-art predictions contain many errors, yet are of great utility and widely-used, we are motivated to design an improved experimental framework for target identification; our method allows the high-throughput assessment of miRNA target sites in a minimally perturbed endogenous cellular environment. The increasingly widespread recognition of the impact of miRNAs on many fields of biology suggests that such efforts could have broad applicability.