Empiric deconvolution of functional RNA elements

描述（由申请人提供）： 信使RNA非翻译区（UTR）在基因表达调控中起着重要作用。许多3'UTR顺式调控元件，包括microRNA靶标和富含AU的元件，已经使用化学交联方法和计算预测鉴定。大多数已知的元素都是从低通量实验中发现的，这些实验速度慢，成本高，而且有很大的偏差。进一步的工作是必要的，以确定新的顺式调节元件，测试计算预测的元件的活性，并发现细胞中3' UTR活性所需的反式调节因子。Erle和McManus实验室一直在合作开发新的工具，用于3' UTR序列的系统功能分析和人类细胞中的全基因组shRNA筛选。我们开发了一种高通量测定法，用于UTR中调控元件的高效大规模平行鉴定（eMPIRE）。eMPIRE检测利用新型四环素调节的慢病毒报告基因构建体、基于大规模平行阵列的寡核苷酸合成、流式细胞术分选和高通量测序。此外，我们开发了一个EXPANDed全基因组慢病毒shRNA文库，使我们能够快速识别UTR调控效应所需的细胞因子。我们 目前的初步数据表明，这些新方法是鉴定人细胞中顺式调节3 ′ UTR序列和反式调节因子的有力工具。没有其他技术能够提供对哺乳动物系统中的功能遗传进行这种定量发现的能力。该项目将产生关于所有人类3'UTR序列的功能活性的全面数据，改进预测3' UTR序列功能活性的计算方法，鉴定新的3' UTR顺式调控元件和基序，并揭示许多在人类细胞中介导3' UTR功能的基因和途径。这是通过两个合作实验室之间的合作而实现的，这两个实验室已经开发了适当的高通量工具，以成功地完成拟议的计划。来自该项目的信息将用于注释基因组数据库，提供对潜在人类多态性的机制性见解。同样，它将为进一步理解由序列元件控制的转录后基因调控奠定令人难以置信的基础。

DESCRIPTION (provided by applicant): Messenger RNA untranslated regions (UTRs) play major roles in regulating gene expression. Many 3' UTR cis-regulatory elements, including microRNA targets and AU-rich elements, have been identified using chemical cross-linking methods and computational predictions. Most of the known elements have been discovered from low throughput experiments, which are slow, expensive, and heavily biased. Further work is necessary to identify novel cis-regulatory elements, test the activity of computationally predicted elements, and discover trans-regulatory factors required for 3' UTR activity in cells. The Erle and McManus laboratories have been working together to generate new tools for systematic functional analysis of 3' UTR sequences and for genome-wide shRNA screens in human cells. We developed a high-throughput assay for efficient Massively Parallel Identification of Regulatory Elements in UTRs (eMPIRE). The eMPIRE assay utilizes a novel tetracycline-regulated lentiviral reporter construct, massively parallel array-based oligonucleotide synthesis, flow cytometric sorting, and high- throughput sequencing. In addition, we developed an EXPANDed genome-wide lentiviral shRNA library that will allow us to rapidly identify cellular factors that are required for UTR regulatory effects. We present preliminary data establishing that these novel methods are powerful tools for identifying cis- regulatory 3' UTR sequences and trans-regulatory factors in human cells. No other technology offers the ability to perform this kind of quantitative discovery for functional genetic in mammalian systems. This project will generate comprehensive data about the functional activity of sequences of all human 3' UTRs, refine computational methods for predicting the functional activity of 3' UTR sequences, identify novel 3' UTR cis-regulatory elements and motifs, and reveal many genes and pathways that mediate 3' UTR functions in human cells. This is made possible via collaboration between two collaborating labs that have developed the appropriate high-throughput tools to successfully accomplish the proposed plan. Information from this project will be used to annotate genomic databases, providing mechanistic insight into potential human polymorphisms. Likewise, it will lay an incredible foundation for further understanding post-transcriptional gene regulation governed by sequence elements.