Molecular mechanisms of translational regulation in aging

衰老转化调控的分子机制

• 批准号: 9564583
• 负责人: Vyacheslav M Labunskyy
• 金额: $ 63.5万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9564583
• 关键词: Address; Affect; Age; Aging; Ally; Applications Grants; Binding; Binding Proteins; Binding Sites; Biological Models; Biotin; Cells; Chimeric Proteins; Complex; Data; Data Set; Elements; Eukaryota; Fluorescence Microscopy; Gene Deletion; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Goals; Human; Individual; Label; Link; Longevity; Magnetism; Mediating; Messenger RNA; Microfluidics; Modeling; Molecular; Molecular Profiling; Monitor; Mothers; Mutation; Organism; Pathway interactions; Pattern; Play; Post-Transcriptional Regulation; Process; Protein Biosynthesis; Proteins; Regulation; Regulatory Element; Reporter; Role; Saccharomycetales; Sorting - Cell Movement; System; Technology; Testing; Transcriptional Regulation; Translating; Translational Regulation; Translations; Work; Yeast Model System; Yeasts; aged; base; cell type; crosslinking and immunoprecipitation sequencing; experimental study; genetic signature; genome-wide; genome-wide analysis; insight; mutant; next generation sequencing; novel; protein function; public health relevance; response; ribosome profiling; tool; transcription factor; transcriptome sequencing

Genome-wide microarray and RNA sequencing studies have revealed changes in the expression of hundreds of genes during aging in diverse organisms. Transcriptional regulation clearly plays an important role in the control of gene expression during aging; however, translation efficiency likely plays an equally important role in determining protein abundance, but has been woefully understudied in this context. Here we propose to study translational changes that are associated with increased longevity and examine the mechanisms of post- transcriptional gene regulation in aging using yeast as a model system. We will test the hypothesis that, in re- sponse to genetic alterations that extend lifespan, mRNA-binding proteins (RBPs) coordinately regulate di- verse cytoprotective genes by affecting their translation efficiency. To identify RBPs involved in regulation of these processes, we will apply RNA-Seq and ribosome profiling combined with next-generation sequencing and characterize transcriptional and translational changes in a panel of long-lived gene deletion mutants identi- fied in genome-wide screens. We propose to integrate translational profiling data obtained for long-lived mu- tants with information about structural and sequence elements recognized by RBPs and build a regulatory in- teraction network. We also propose to carry out ribosome profiling in replicatively aged wild-type cells and long-lived mutant strains to globally identify genes whose expression is affected by translational regulation dur- ing aging. Finally, we will utilize cutting-edge microfluidic technologies to validate and extend these discover- ies at the single-cell level. Comparing translational profiles in young and replicatively aged wild-type yeast and multiple long-lived deletion mutants will reveal genetic signatures associated with increased longevity and will allow us to identify novel RBPs involved in translational regulation during aging. We will then characterize RBPs and directly identify their mRNA-binding targets using CLIP-Seq. These data will allow us to uncover specific mechanisms and identify cis-regulatory elements that are responsible for translational changes ob- served in long-lived mutants. We will also use fluorescence microscopy and microfluidic cell trapping in order to monitor how the abundance of RBPs changes with age in individual mother cells. Finally, we will test if the candidate RBPs identified from CLIP-Seq and microfluidics experiments play a causal role in mediating the lifespan extension through genetic epistasis analysis in order to determine whether candidate RBPs are nec- essary and sufficient for lifespan extension. Successful completion of this study will add valuable insight into translational regulation of aging, and may provide a better understanding of the molecular mechanisms that regulate aging in humans.

全基因组微阵列和RNA测序研究揭示了数百种表达的变化 不同生物体衰老过程中的基因。转录调节显然在 在衰老过程中控制基因表达；但是，翻译效率可能在 确定蛋白质的丰度，但在这种情况下已被严重研究。在这里我们建议学习 转化变化与寿命的增加相关并检查后的机制 使用酵母作为模型系统的衰老中的转录基因调节。我们将检验以下假设 对延长寿命，mRNA结合蛋白（RBP）的遗传改变的响应 经文细胞保护基因通过影响其翻译效率。确定参与调节的RBP 这些过程，我们将应用RNA-Seq和核糖体分析，并结合下一代测序 并表征了一系列长寿命缺失突变体鉴定的转录和翻译变化 在全基因组筛选中融合。我们建议整合为长寿命MU-获得的翻译分析数据 带有RBP认可的结构和序列元件的信息，并建立一个调节性的内部 Teraction网络。我们还建议在复制老化的野生型细胞和 长寿命突变菌株以鉴定其表达受翻译调节影响的基因 老化。最后，我们将利用尖端的微流体技术来验证和扩展这些发现 - IE在单细胞级别。比较年轻和复制的野生型酵母和复制的翻译轮廓和 多个长期寿命的缺失突变体将揭示与寿命增加相关的遗传特征，并将 允许我们确定衰老期间参与翻译调节的新型RBP。然后我们将表征 RBP并直接使用夹子序列直接识别其mRNA结合靶标。这些数据将使我们能够发现 具体机制并确定负责转移变化的顺式调节元素 在长寿突变体中服役。我们还将按顺序使用荧光显微镜和微流体细胞捕获 监测单个母细胞中RBP的丰度随着年龄的增长而变化。最后，我们将测试是否 从夹子seq和微流体实验中鉴定出的候选RBP在介导 通过遗传上毒分析延长寿命，以确定候选RBP是否为NEC- 精致且足以延长寿命。这项研究的成功完成将增加对 衰老的翻译调节，并可以更好地理解分子机制 调节人类衰老。