Dissecting Translational Regulation by Genome-Wide Mapping of Initiation Factors

描述(由申请人提供)：适当的基因表达控制对于几乎所有的生物过程都是重要的，从发育到肿瘤发生。转录后调控是控制基因表达的一个重要机制，它通常是通过信使RNA本身的特定序列来调节的，通常会导致mRNA稳定性的变化和/或翻译起始的调节。一种重要的转录后调控机制是由microRNAs(MiRNAs)介导的。这些小RNA识别靶mRNA中的互补序列，主要通过mRNA失稳和抑制翻译启动来抑制基因表达。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，翻译启动机制如何在转录组范围内结合mRNAs，以及这种相互作用受到调控的程度仍未确定。长期以来，我一直对转录后调控机制感兴趣。在我与克里斯·诺伯里博士一起在牛津大学攻读研究生期间，我发现了裂殖酵母中一种未知的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miRNAs的降解。虽然大多数miRNAs是稳定的，但我发现扩展的miR-16家族中有几个成员异常不稳定，并进一步表明这种不稳定使得该家族能够在细胞周期中进行动态调节。这项工作是由Ruth L.Kirschstein NRSA奖学金资助的。我的长期目标是了解mRNAs和调控因子之间的相互作用，并定义这些相互作用如何在转录组范围内调节基因表达。作为一家学术机构的研究小组组长，我想继续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)确定翻译启动过程中受调控过程调控的步骤(S)；2)接受额外科学技能的培训，特别是计算生物学领域的培训，以便我能够作为一名独立的研究人员取得成功。这项拟议研究的基本原理是，翻译起始因子结合位点的转录组范围的图谱将提供对调节机制的洞察和 将同时使我能够发展计算技能。这里提出的工作包括两个目标。在目标1，将在指导阶段完成，我将研究miRNAs抑制翻译启动的分子机制(S)，这是一个仍然未知和有争议的话题。为了验证我的中心假设，即miRNA介导的翻译抑制通过破坏eIF4G结合来发挥作用，我将在整个转录范围内绘制eIF4G-mRNA的相互作用图，并确定这些相互作用是如何受到miRNAs的影响的。作为这一重要问题的一种新方法，拟议的工作将在以前仅限于机制研究的水平上分析miRNA介导的抑制的影响，但在转录组范围的方法的规模上。重要的是，这个目标将使我能够发展实验和计算技能，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，巴特尔实验室是此次培训的理想场所。在……里面 目标2，我将在指导阶段启动，并在独立阶段完成，我将扩展这项工作，以在转录组范围内绘制其他启动因子的结合图谱。假设调控经常通过不同的、可控的启动因子结合发生，我将绘制其他因子的结合图，如eIF4E和eIF4A，并确定这些相互作用如何与翻译效率相关。我还将把这些分析应用于天然的非翻译转录本，即细胞质中的长插入非编码RNA(LincRNAs)，从而阐明取消翻译的具体机制。一旦整合，这些结果将提供一个新的视角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培训，以及在高通量测序和基本计算分析方面的经验。我在转录后调控方面的实验背景和专业知识使我能够胜任拟议的工作。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，这些方法将成为我独立研究的基础，并将为我作为独立调查员开始职业生涯提供平台。 与公共健康相关：尽管所有细胞都有相同的基因集合，但它们的不同之处在于哪些基因被打开，哪些被关闭。当基因表达不当时，癌症有时会发展和/或生物体的发育可能不适当地发生。这项提议的重点是全面了解启动基因的最后一步--即制造蛋白质产物--是如何控制的。

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.