Dissecting Translational Regulation by Genome-Wide Mapping of Initiation Factors

描述（由申请人提供）：基因表达的适当控制对几乎所有生物学过程都很重要，从发育到肿瘤发生。控制基因表达的一个重要机制是转录后调节，其通常通过信使RNA（mRNA）本身中的特定序列介导，并且通常导致mRNA稳定性的变化和/或翻译起始的调节。一个突出的转录后调节机制是由microRNA（miRNAs）介导的。这些小RNA识别靶mRNA中的互补序列并抑制基因表达，主要通过mRNA不稳定以及抑制翻译起始。然而，miRNAs介导翻译起始抑制的分子机制尚不清楚。更广泛地说，翻译起始机制如何在转录组范围内结合mRNA，以及这种相互作用的调节程度仍然没有得到表征。 我对转录后调控机制有着长期的兴趣。在牛津大学与克里斯·诺伯里博士（Chris Norbury）的研究生学习期间（由罗兹奖学金资助），我在粟酒裂殖酵母中发现了一条未知的mRNA衰变途径。然后，作为怀特黑德研究所大卫巴特尔博士实验室的博士后研究员，我继续研究RNA衰变途径，并研究了miRNA的降解。虽然大多数miRNAs是稳定的，但我发现了扩展的miR-16家族的几个成员异常不稳定，并进一步表明这种不稳定性使该家族能够在细胞周期中进行动态调节。这项工作是由一个露丝L。Kirschstein NRSA奖学金。 我的长期目标是了解mRNA和调控因子之间的相互作用，并确定这些相互作用如何反过来在转录组范围内调节基因表达。我想作为一个学术机构的研究小组的负责人来研究这个令人兴奋的话题。为了实现这一目标，本申请的总体目标是：1）确定由调节过程调节的翻译起始步骤; 2）接受额外科学技能的培训，特别是在计算生物学领域，以便我能够成功地作为独立研究者。这项研究的基本原理是，对翻译起始因子结合位点的转录组范围作图将提供对调控机制的深入了解， 同时也能让我发展计算能力。 这里提出的工作包括两个目标。在目标1中，将在指导阶段完成，我将研究miRNAs抑制翻译起始的分子机制，这是一个仍然未知和有争议的话题。为了验证我的核心假设，即miRNA介导的翻译抑制通过破坏eIF 4G结合起作用，我将在转录组范围内绘制eIF 4G-mRNA相互作用，并确定这些相互作用如何受到miRNA的影响。一种新的方法来解决这个重要的问题，拟议的工作将在以前仅限于机制研究的水平上，但在转录组范围内的方法的规模上测定miRNA介导的抑制的影响。重要的是，这一目标将使我能够发展实验和计算技能，用于在翻译的背景下研究蛋白质-RNA相互作用。凭借其在miRNA生物学方面的长期经验，深厚的计算专业知识和持续的技术创新，Bartel实验室是这种培训的理想场所。在 目标2，我将在指导阶段开始，并在独立阶段完成，我将扩展这项工作，以映射在转录组范围内的其他启动因子的结合。假设调节经常发生通过差异，控制结合的起始因子，我将映射其他因素的结合，如eIF 4 E和eIF 4A，并确定这些相互作用如何与翻译效率。我还将这些分析应用于自然非翻译转录本，细胞质长插入非编码RNA（lincRNA），从而阐明废除翻译的具体机制。一旦整合，这些结果将提供一个新的角度来考虑转录后基因调控。 通过我以前的工作，我有广泛的分子生物学训练以及高通量测序和基本计算分析的经验。我在转录后调控方面的实验背景和专业知识使我能胜任这项工作.本提案中所述的培训和研究将使我能够开发一套方法工具包，这些方法将构成我独立研究的基础，并将为我作为独立调查员开展职业生涯提供平台。 公共卫生相关性：虽然所有细胞都有相同的基因集合，但使它们不同的是哪些基因被打开，哪些基因被关闭。当基因不适当地表达时，癌症有时会发展和/或生物体的发育可能会不适当地发生。这项提案的重点是全面了解如何在打开一个基因的最后一步，也就是说，使蛋白质产品，是控制。

DESCRIPTION (provided by applicant): Proper control of gene expression is important for nearly all biological processes, ranging from development to oncogenesis. One important mechanism for controlling gene expression is post-transcriptional regulation, which is normally mediated through specific sequences in the messenger RNA (mRNA) itself and often results in changes in mRNA stability and/or modulation of translational initiation. One prominent post-transcriptional regulatory mechanism is that mediated by microRNAs (miRNAs). These small RNAs recognize complementary sequences in a target mRNA and repress gene expression, primarily through mRNA destabilization as well as by inhibition of translation initiation. Nevertheless, the molecular mechanism by which miRNAs mediate repression of translation initiation is unclear. More broadly, how the translation initiation machinery binds mRNAs on a transcriptome-wide scale and the extent to which such interactions are regulated remain uncharacterized. I have had a long-standing interest in post-transcriptional regulatory mechanisms. During my graduate studies at the University of Oxford with Dr. Chris Norbury, which were funded by a Rhodes Scholarship, I uncovered an unknown mRNA decay pathway in Schizosaccharomyces pombe. Then, as a post-doctoral fellow in the laboratory of Dr. David Bartel at the Whitehead Institute, I continued to investigate RNA decay pathways and studied the degradation of miRNAs. Although the majority of miRNAs are stable, I identified several members of the extended miR-16 family as unusually unstable, and furthermore showed that this instability enabled dynamic regulation of the family in the cell cycle. This work was funded by a Ruth L. Kirschstein NRSA fellowship. My long-term goal is to understand interactions between mRNAs and regulatory factors and to define how these interactions in turn modulate gene expression on a transcriptome-wide scale. I would like to pursue this exciting topic as the leader of a research group in an academic institution. To achieve this goal, the overall objectives of this application are: 1) to identify the step(s) in translation initiation that are modulated by regulatory processes; 2) to receive training in additional scientific skills, especialy in the area of computational biology, so that I can be successful as an independent investigator. The rationale that underlies the proposed research is that transcriptome-wide mapping of the binding sites of translation initiation factors will provide insight into regulatory mechanisms and will simultaneously enable me to develop computational skills. The work proposed here comprises two aims. In Aim 1, which will be completed during the mentored phase, I will investigate the molecular mechanism(s) by which miRNAs repress translation initiation, a topic that remains unknown and controversial. To test my central hypothesis that the miRNA-mediated translational repression acts by disrupting eIF4G binding, I will map eIF4G-mRNA interactions transcriptome-wide and determine how these interactions are affected by miRNAs. A novel approach to this important issue, the proposed work will assay effects of miRNA-mediated repression on a level formerly restricted to the mechanistic studies but on the scale of the transcriptome-wide approaches. Importantly, this aim will allow me to develop experimental and computational skills for studying protein-RNA interactions transcriptome-wide in the context of translation. With its long experience in miRNA biology, deep computational expertise and consistent technological innovation, the Bartel laboratory is the ideal place for this training. In Aim 2, which I will initiate during the mentored phase and complete in the independent phase, I will extend this work to map binding of other initiation factors on a transcriptome-wide scale. Hypothesizing that regulation often occurs through differential, controlled binding of initiation factors, I will map the binding of other factors, such as eIF4E and eIF4A, and determine how these interactions relate to translational efficiency. I will also apply these analyses to naturall non-translated transcripts, cytoplasmic long intervening non-coding RNAs (lincRNAs), and thereby shed light on specific mechanisms abrogating translation. Once integrated, these results will provide a new perspective from which to consider post-transcriptional gene regulation. Through my previous work, I have extensive molecular biology training as well as experience in high- throughput sequencing and basic computational analysis. My experimental background and expertise in post- transcriptional regulation ideally positions me for the proposed work. The training and research described in this proposal will allow me to develop a tool-kit of approaches that will form the basis of my independent research, and will provide the platform from which to launch my career as an independent investigator. PUBLIC HEALTH RELEVANCE: Although all cells have the same collection of genes, what makes them different is which genes are turned on and which are turned off. When genes are inappropriately expressed, cancer can sometimes develop and/or the development of an organism can occur improperly. This proposal focuses on comprehensively understanding how the final step in turning on a gene,-that is, making the protein product,-is controlled.