Regulatory Mechanisms and Biological Functions of non-coding RNA

描述（由申请人提供）：最近分析真核转录组的工作揭示了广泛的非编码RNA（ncRNA）表达的惊人观察结果。在整个基因组中普遍发现，一些ncRNA，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。与这一观点一致，已经发现ncRNA能够显著改变基因表达模式，有时通过招募染色质修饰酶。还发现ncRNA在整个真核生物中高度保守。虽然，总的来说，这都表明ncRNA发挥重要的生物学作用，但绝大多数ncRNA的功能仍然未知。一份这样的报告在芽殖酵母（酿酒酵母）中鉴定出约1,000种ncRNA，它们来源于基因间（基因间）和基因内（基因内）， 在正义和反义方向，其中大多数具有完全未知的功能。此外，尽管选择ncRNA的作用已得到充分证实，但对ncRNA的调控知之甚少。最近，ncRNA的错误调节与小鼠中肿瘤转移的发生率增加有关。这项题为“非编码RNA的调控机制和生物学功能”的提案旨在阐明ncRNA调控背后的分子基础，以及可能成为其广泛表达基础的任何生物学功能。鉴于它们与肿瘤转移的相关性，不能低估对它们的生物学功能和调节的理解。该提案主要采用分子遗传学技术：1）揭示ncRNA负调控mRNA表达的机制，2）在芽殖酵母（酿酒酵母）中鉴定ncRNA转录的全基因组抑制因子。为了实现第一个目标，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mRNA似乎受到重叠ncRNA的负调控，称为转录干扰。随后的基因本体论（GO）分析发现了几种参与生长调节的途径，这种调节机制似乎是常用的。我们将系统地对这些基因进行排序，并采用一系列遗传结构来揭示这些基因受到负调控的机制。在后者的目标，我们进行了系统的遗传分析，以确定，在一个高通量和公正的方式，308推定的ncRNA阻遏物。S.酿酒酵母的遗传易处理性使它特别适合这样的筛选， 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ncRNA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。 公共卫生相关性：非编码RNA（ncRNA）与关键的发育过程（如X染色体失活）和疾病状态（如癌症）有关，这强调了了解其生物学功能和调控机制的日益增长的需求。一些报道表明，参与肿瘤转移的ncRNA能够广泛改变基因表达模式。该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疾病状态转变的线索，可能揭示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. )