RNA dynamics and co-translational decay in aging and aging-related disease

衰老和衰老相关疾病中的 RNA 动力学和共翻译衰减

• 批准号: 10252559
• 负责人: Emmanouil Maragkakis
• 金额: $ 168.48万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10252559
• 关键词: APP-PS1; Affect; Age; Aging; Alzheimer' s Disease; Biological; Biological Process; CD19 gene; Cell physiology; Cells; Complex; Coupled; DNA; Data; Data Set; Defect; Development; Disease; Encapsulated; Frameshift Mutation; Genes; Genetic Transcription; Genome; Genomics; Hela Cells; Homeostasis; Introns; Intuition; Libraries; Machine Learning; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Mitochondria; Modeling; Molecular; Morphology; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Phase; Presenile Alzheimer Dementia; Process; Property; Proteins; Protocols documentation; Publishing; RNA; RNA Decay; RNA Splicing; RNA-Binding Proteins; Resistance; Ribosomes; Techniques; Tissues; Training; Transgenic Mice; Translations; Work; cohort; computerized tools; crosslinking and immunoprecipitation sequencing; genomic data; human RNA sequencing; leukemia; mRNA Decay; mRNA Transcript Degradation; mouse model; new technology; novel; open source; programs; transcriptome; transcriptome sequencing; transcriptomics

While genomes encapsulate the information for the development and function of cells most of the biological complexity lies on high-order interactions that manifest at the DNA, RNA and protein level. The understanding of these multifaceted regulatory mechanisms is of paramount importance for the understanding of complex, polygenic processes such as ageing. In previous works we described a co-translational pathway of mRNA decay that is mediated by ribosome-phased cleavage of the mRNA (Ibrahim et al., 2018). We developed a new protocol, Akron-Seq, and integrated orthogonal large-scale genomic datasets to identify that the process is initiated by a ribosome stall that triggers an endonucleolytic cleavage and results in propagating cleavages by upstream ribosomes. Published protocols such as 5P-Seq or Akron-Seq are able to capture native mRNA ends but not both ends on the same molecule, therefore limiting our ability to probe mRNA properties and molecular pathways that affect both ends of the mRNA molecules. Our group is developing and using new, combined experimental/computational approaches to capture the native ends of mRNA molecules in cells and tissues to profile RNA in unprecedented detail. We have initially successfully applied these techniques involving long-read sequencing in HeLa cells to establish and optimize the protocols. We have also developed new computational tools for the analysis of the novel datasets. One of our computational tools (SamQL) has been packaged and offered as an open-source library and executable program for intuitive and complex queries on high-throughput data stored in SAM/BAM files. By analyzing our novel long-read sequencing data and integrating orthogonal CLIP-Seq datasets we have discovered a group of RNA-binding proteins that are potentially associated with the control of co-translational RNA decay and that have been implicated with cancer and neurodegenerative disease. Similar to our previous work, where we identify the effect of an ALS-associated mutation in gene translation and mitochondria morphology (Nakaya and Maragkakis, 2018), we are following up on these discoveries to dissect the molecular mechanisms guided by the identified proteins. Additionally, we are analyzing, computationally, large-scale transcriptomic data from studies such as the GESTALT to discover age-associated changes in RNA dynamics and the ratio between RNA transcription and decay. Moreover, we have initiated a pilot analysis of the transcriptomic profile of aging APP/PS1 double transgenic mice, associated with early-onset Alzheimer's disease. Finally, by using long-read transcriptome sequencing we have found that the retention of intron 2 in CD19 contributes to CART-19 resistance in leukemias with CD19 subclonal frameshift mutations (Asnani et al., 2020).

虽然基因组封装了细胞发育和功能的信息，但大多数生物学复杂性都取决于在DNA，RNA和蛋白质水平上表现出来的高阶相互作用。了解这些多方面的调节机制对于理解复杂的多基因过程（如衰老）至关重要。 在先前的工作中，我们描述了由mRNA的核糖体相切割介导的mRNA衰变的共翻译途径（Ibrahim et al.，2018年）。我们开发了一种新的协议，Akron-Seq，并整合了正交的大规模基因组数据集，以确定该过程是由核糖体失速引发的，该核糖体失速引发内切核酸裂解并导致上游核糖体的增殖裂解。已发表的方案如5 P-Seq或Akron-Seq能够捕获天然mRNA末端，但不能捕获同一分子上的两个末端，因此限制了我们探测影响mRNA分子两端的mRNA性质和分子途径的能力。我们的团队正在开发和使用新的，结合实验/计算的方法来捕获细胞和组织中mRNA分子的天然末端，以前所未有的细节描述RNA。 我们已经初步成功地应用了这些技术，涉及HeLa细胞中的长读序，以建立和优化协议。我们还开发了新的计算工具来分析新的数据集。我们的计算工具之一（SamQL）已被打包并作为开源库和可执行程序提供，用于对存储在SAM/BAM文件中的高吞吐量数据进行直观和复杂的查询。通过分析我们新的长读段测序数据并整合正交CLIP-Seq数据集，我们发现了一组RNA结合蛋白，它们可能与控制共翻译RNA衰变相关，并与癌症和神经退行性疾病有关。与我们以前的工作类似，我们确定了ALS相关突变对基因翻译和线粒体形态的影响（Nakaya和Maragkakis，2018），我们正在跟进这些发现，以剖析由所确定的蛋白质指导的分子机制。此外，我们正在通过计算分析来自GESTALT等研究的大规模转录组数据，以发现RNA动力学中与年龄相关的变化以及RNA转录和衰变之间的比率。此外，我们已经开始了一个试点分析的转录谱老化APP/PS1双转基因小鼠，与早发性阿尔茨海默病。最后，通过使用长读段转录组测序，我们已经发现，在具有CD 19亚克隆移码突变的白血病中，内含子2在CD 19中的保留有助于CART-19抗性（Asnani等人，2020年）。