Dissecting Translational Regulation by Genome-Wide Mapping of Initiation Factors

通过启动因子的全基因组图谱剖析翻译调控

基本信息

批准号：
8538468
负责人：
Olivia Selfridge Rissland
金额：
$ 8.76万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8538468
关键词：
Affect Area Binding Binding Sites Biological Biological Assay Biological Process Biology Cell Cycle Cells Collection Computational Biology Computational Technique Computer Analysis Data Set Defect Development Disease Family Fellowship Fibrinogen Fission Yeast Functional RNA Funding Gene Expression Gene Expression Profile Gene Expression Regulation Genes Goals In Vitro Institutes Institution Laboratories Learning Light Malignant Neoplasms Maps Mediating Mentors Messenger RNA MicroRNAs Modeling Molecular Molecular Biology Monitor National Research Service Awards Organism Pathway interactions Peptide Initiation Factors Phase Positioning Attribute Post-Transcriptional Regulation Postdoctoral Fellow Process Proteins RNA Decay RNA Stability RNA-Protein Interaction Regulation Repression Research Research Personnel Research Training Scholarship Small RNA Techniques Testing Training Transcript Translation Initiation Translational Regulation Translational Repression Translations Universities Work base carcinogenesis career experience genetic regulatory protein genome-wide insight interest member novel strategies skills success technological innovation tool treatment effect tumorigenesis

项目摘要

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.

描述（由申请人提供）：基因表达的正确控制对于几乎所有生物过程（从发育到肿瘤发生）都很重要。控制基因表达的一种重要机制是转录后调节，它通常通过信使 RNA (mRNA) 本身的特定序列介导，并且常常导致 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 microRNA (miRNA) 介导的。这些小 RNA 识别靶标 mRNA 中的互补序列并主要通过 mRNA 不稳定以及抑制翻译起始来抑制基因表达。然而，miRNA 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 以及这种相互作用受到调节的程度仍然未知。我长期以来对转录后调控机制很感兴趣。在罗德奖学金资助下，我在牛津大学跟随 Chris Norbury 博士进行研究生学习期间，我发现了粟酒裂殖酵母中未知的 mRNA 衰变途径。然后，作为怀特海研究所 David Bartel 博士实验室的博士后研究员，我继续研究 RNA 衰减途径并研究 miRNA 的降解。尽管大多数 miRNA 是稳定的，但我发现 miR-16 扩展家族的几个成员异常不稳定，并且进一步表明这种不稳定性使得该家族在细胞周期中能够动态调节。这项工作由 Ruth L. Kirschstein NRSA 奖学金资助。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在转录组范围内调节基因表达。我想作为学术机构研究小组的领导者来研究这个令人兴奋的课题。为了实现这一目标，该应用程序的总体目标是： 1) 识别翻译启动中受监管流程调节的步骤； 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，以便我能够成为一名成功的独立研究者。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图谱将提供对调节机制和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）中，我将研究 miRNA 抑制翻译起始的分子机制，这是一个仍然未知且有争议的话题。为了检验我的中心假设，即 miRNA 介导的翻译抑制通过破坏 eIF4G 结合来发挥作用，我将在转录组范围内绘制 eIF4G-mRNA 相互作用图，并确定这些相互作用如何受到 miRNA 的影响。拟议的工作是解决这一重要问题的一种新方法，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，但在转录组范围内的方法的规模上进行分析。重要的是，这个目标将使我能够发展实验和计算技能，以研究翻译背景下转录组范围内的蛋白质-RNA 相互作用。凭借在 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 实验室是此次培训的理想场所。在目标 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。假设调节通常通过起始因子的差异性、受控结合发生，我将绘制其他因子（例如 eIF4E 和 eIF4A）的结合图，并确定这些相互作用与翻译效率的关系。我还将这些分析应用于天然非翻译转录本、细胞质长间插非编码 RNA (lincRNA)，从而阐明废除翻译的特定机制。一旦整合，这些结果将为考虑转录后基因调控提供新的视角。通过我之前的工作，我接受了广泛的分子生物学培训以及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为我所提出的工作奠定了理想的基础。本提案中描述的培训和研究将使我能够开发出一套方法工具包，该工具包将构成我的独立研究的基础，并为我作为独立调查员开启职业生涯提供平台。