Regulatory Mechanisms and Biological Functions of non-coding RNA

描述(由申请人提供)：最近对真核细胞转录组的分析揭示了令人惊讶的观察到广泛的非编码RNA(NcRNA)表达。在整个基因组中发现的一些ncRNAs，如Xist和HOTAIR，已被发现分别参与关键的发育过程和疾病状态，如X染色体失活和癌症。与这一概念一致，已发现ncRNAs能够显著改变基因表达模式，有时是通过招募染色质修饰酶。NcRNAs在真核生物中也被发现是高度保守的。总体而言，所有这些都表明ncRNAs发挥着重要的生物学作用，但绝大多数ncRNAs的功能仍然未知。一份这样的报告在萌芽酵母中发现了大约1000个ncRNAs，它们隐蔽地起源于基因间(基因间)和基因内(基因内)，以及 在正义和反义定向中，大多数具有完全未知的功能。此外，尽管特定的ncRNA的作用已经确立，但对ncRNA的调控知之甚少。最近，ncRNAs的错误调控与小鼠肿瘤转移发生率的增加有关。这项题为“非编码RNA的调控机制和生物学功能”的提案试图阐明ncRNA调控背后的分子基础，以及可能支撑其广泛表达的任何生物学功能。考虑到它们与肿瘤转移的相关性，了解它们的生物学功能和调控是不能低估的。该建议主要利用分子遗传学技术：1)揭示ncRNAs负性调控mRNA表达的机制；2)在萌芽酵母中发现ncRNA转录的全基因组抑制因子。对于第一个目标，使用微阵列分析，我们已经在整个基因组中找到了候选基因，其mRNAs似乎受到重叠的ncRNA的负调控，称为转录干扰。随后的基因本体论(GO)分析发现了几条参与生长调控的途径，在这些途径中，这种调控机制似乎得到了普遍使用。我们将系统地对这些基因进行排序，并使用一系列基因结构来揭示这些基因变得负调控的机制。在后一个目标中，我们进行了系统的遗传分析，以高通量和无偏见的方式鉴定了308个可能的ncRNA抑制因子。酿酒酵母的遗传易感性使其特别容易受到这样的筛选， 这是以前没有开发过的。最终，我们希望这项提案中概述的工作能够阐明ncRNAs如何能够促进不同的基因表达模式，这可以揭示人类疾病状态的分子基础。 与公共卫生相关：非编码RNA(NcRNA)与X染色体失活等关键发育过程和癌症等疾病状态有关，突显出越来越需要了解它们的生物学功能和调节机制。一些报道表明，参与肿瘤转移的ncRNAs能够广泛改变基因表达模式。这项提案中概述的工作将提供更多关于ncRNA物种如何影响基因调控和影响向疾病状态转变的线索，可能揭示一个可用于长期诊断和预后的特征。)

DESCRIPTION (provided by applicant): Recent work analyzing the eukaryotic transcriptome revealed the surprising observation of widespread, non- coding RNA (ncRNA) expression. Found globally throughout the genome, a few ncRNAs, such as Xist and HOTAIR, have been found to be involved in key developmental processes and disease states, such as X- chromosome inactivation and cancer, respectively. Consistent with this notion, ncRNAs have been found to be capable of dramatically altering gene expression patterns, sometimes by the recruitment of chromatin modifying enzymes. ncRNAs have also been found to be heavily-conserved throughout eukaryotes. While, collectively, this all suggests that ncRNAs play significant biological roles, the functions of the vast majority of ncRNAs are still unknown. One such report identified ~1,000 ncRNAs in the budding yeast, Saccharomyces cerevisiae, originating cryptically, both intergenically (between genes) and intragenically (within genes), and in the sense and anti-sense orientations, the majority of which have entirely unknown functions. Furthermore, even less is known of ncRNA regulation, despite the well-established roles of select ncRNAs. More recently, misregulation of ncRNAs has been associated with increased incidence of tumor metastasis in mice. This proposal, entitled "Regulatory Mechanisms and Biological Functions of non-coding RNA," seeks to elucidate the molecular underpinnings behind the regulation of ncRNAs, as well as any biological functions that might underlie their widespread expression. Given their relevance to tumor metastasis, understanding their biological function and regulation cannot be understated. The proposal mainly employs molecular genetic techniques to: 1) uncover the mechanism by which ncRNAs negatively regulates mRNA expression and 2) identify genome-wide suppressors of ncRNA transcription, in the budding yeast, Saccharomyces cerevisiae. Towards the first aim, using microarray analysis, we already have candidate genes throughout the genome whose mRNAs seems to be negatively regulated by the overlapping ncRNA, termed transcriptional interference. Subsequent Gene Ontology (GO) analysis uncovered several pathways involved in growth regulation where this kind of regulatory mechanism seems to be commonly used. We will systematically rank these genes, and employ a series of genetic constructs to uncover the mechanism by which these genes become negatively regulated. In the latter aim, we have performed systematic genetic analysis to identify, in a high-throughput and unbiased manner, 308 putative ncRNA repressors. S. cerevisiae's genetic tractability make it especially amenable to a screen like this, which has not been previously developed. Ultimately, we would like the work outlined in this proposal to shed light on how ncRNAs are capable of promoting distinct gene expression patterns, which can reveal the molecular basis underlying human disease states. PUBLIC HEALTH RELEVANCE: Non-coding RNAs (ncRNA) have been implicated in key developmental processes, such as X-chromosome inactivation, and disease states, such as cancer, underscoring the growing need to understand their biological functions and regulatory mechanisms. Several reports suggest that ncRNAs involved in tumor metastasis are capable of widespread alteration of gene expression patterns. The work outlined in this proposal will provide more clues as to how ncRNA species might affect gene regulation and influence the transition to disease states, potentially revealing a signature that can be used in diagnosis and prognosis in the long-term. )