Illuminating the immune system's genomic dark matter: functionally annotating the hidden translatome

照亮免疫系统的基因组暗物质：对隐藏的翻译组进行功能注释

• 批准号: 10245900
• 负责人: Ruaidhri Jackson
• 金额: $ 152.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245900
• 关键词: Acceleration; Adherence; Animal Model; Back; Biological Process; Chimeric Proteins; Classification; Code; Disease; Event; Foundations; Gene Silencing; Genes; Genome; Genomics; Human; Immune response; Immune system; Immunity; Inflammation; Inflammatory Response; Investigation; Messenger RNA; Open Reading Frames; Physiological; Physiology; Process; Protein Biosynthesis; Proteins; RNA; RNA Splicing; Resolution; Role; Technology; Testing; Trans-Splicing; Transcript; Translations; Untranslated RNA; Work; dark matter; differential expression; gain of function; genome-wide; human disease; innovation; knock-down; macrophage; mammalian genome; new therapeutic target; novel; ribosome profiling; screening; transcriptomics; translatome

Project Summary/Abstract: The annotation of the mammalian protein coding genome is alarmingly incomplete. Despite the massive acceleration and widespread use of transcriptomic approaches to understand biological processes, we still do not fundamentally understand RNA translation at its most basic level. Traditional definitions that categorize an RNA as either protein coding or non-coding, are currently incompatible with recent findings from genome wide translatome studies. We hypothesize that a combination of technological barriers and an adherence to dogmatic assumptions of what constitutes an open reading frame (ORF) and protein coding RNA, have severely constrained identification of a plethora of novel regulators of biological processes. We term this uncharacterized material genomic dark matter. In this proposal, we aim to systematically identify and functionally uncover its true contribution to the inflammatory response. By utilizing ribosome profiling in steady state and activated macrophages, we have identified “non-coding” RNA undergoing robust translation. Furthermore, we uncovered widespread polycistronic translation of multiple ORFs within classically annotated protein coding genes. To reveal the functional contribution of these alternative ORFs and delineate their role from that of their gene’s annotated ORF, we propose to conduct a parallel loss and gain of function inverse screening approach to identify novel proteins that contribute to the inflammatory response. Furthermore, although protein coding genes are thought to solely function by providing a message for protein synthesis, we have identified a class of mRNAs that are highly differentially expressed following bacterial stimulation but are not translated. This philosophically questions the very classification of a protein coding gene. By combining transcriptional silencing and ORF disruption studies, we aim to decouple the functional contribution of a gene’s RNA from its coding potential. In addition, although current ribosome profiling technologies are ill suited to discover unannotated transcripts undergoing translation, we have identified a plethora of mRNA derived from atypical “trans-splicing” between two different transcripts that appear to encode novel chimeric proteins. By developing an innovative technological pipeline, RiboFusionSeq, we will generate the first rigorous identification platform for coding chimeric RNAs. Furthermore, to uncover the capacity of intra-transcript circularization via “back-splicing” to encode novel protein, RiboCircSeq will be established. Using knockdown approaches specifically targeting trans- and back-splicing events, we will conduct the first functional screening of proteins derived from atypical splicing and interrogate their contribution to immunity. Finally, we will generate animal models to test the mechanistic and physiological importance of our findings in inflammation and disease. Together, these studies will 1) provide a transformative level of resolution on the protein coding genome during the immune response, 2) establish a new paradigm for the functional annotation of mammalian genomes, 3) identify a plethora of new molecules for further investigation and 4) have far reaching implications to understanding the processes underlying all human diseases.

项目摘要/摘要： 哺乳动物蛋白质编码基因组的注释令人震惊地不完整。尽管很大 转录组方法的加速和广泛使用以了解生物学过程，我们仍然会这样做 从根本上不理解最基本的RNA翻译。传统定义将 RNA作为蛋白质编码或非编码，目前与基因组范围的最新发现不相容 翻译组研究。我们假设技术障碍和遵守教条的结合 构成开放阅读框（ORF）和蛋白质编码RNA的假设严重 对许多生物过程的新调节剂的限制鉴定。我们称这个未示威 物质基因组暗物质。在此提案中，我们旨在系统地识别并在功能上揭示其真实性 对炎症反应的贡献。通过在稳态下使用核糖体分析并激活 巨噬细胞，我们已经确定了正在进行稳健翻译的“非编码” RNA。此外，我们发现了 经典注释的蛋白质编码基因中多个ORF的宽度多源翻译。到 揭示这些替代ORF的功能贡献，并从其基因的角色中划定了它们的作用 带注释的ORF，我们建议进行平行的损失和功能相反筛选方法的增益，以识别 有助于炎症反应的新型蛋白质。此外，尽管蛋白质编码基因是 我们认为仅通过提供蛋白质合成的消息来发挥作用，我们已经确定了一类mRNA 在细菌刺激后表达的高度不同，但没有翻译。从哲学上讲 质疑蛋白质编码基因的分类。通过结合转录沉默和ORF 破坏研究，我们旨在将基因RNA的功能贡献与其编码潜力相结合。在 尽管当前的核糖体分析技术不适合发现未注释的成绩单 经过翻译，我们已经确定了源自非典型“跨性”的众多mRNA 似乎编码新型嵌合蛋白的两个不同转录本。通过开发创新的技术 管道，RiboFusionSeq，我们将生成第一个用于编码嵌合RNA的严格标识平台。 此外，要通过“背拼图”编码新蛋白质的转录内电路的能力， 将建立Ribocircseq。使用专门针对反式和后拼图的敲低方法 事件，我们将对源自非典型剪接和询问的蛋白质进行第一次功能筛选 他们对免疫的贡献。最后，我们将生成动物模型来测试机械和生理 我们在炎症和疾病中发现的重要性。这些研究将共同​​提供1）提供变革性的 免疫响应期间蛋白质编码基因组的分辨率水平，2）建立一个新的范式 哺乳动物基因组的功能注释，3）识别大量新分子以进行进一步研究 4）具有了解所有人类疾病基础的过程的影响很大。