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.

项目概要/摘要： 哺乳动物蛋白质编码基因组的注释是惊人的不完整。尽管有大量的 加速和广泛使用转录组学方法来理解生物过程，我们仍然这样做 不能从根本上理解RNA翻译的最基本的水平。传统的定义， RNA作为蛋白质编码或非编码，目前与全基因组的最新发现不一致 翻译研究我们假设，技术障碍和坚持教条主义的结合 关于什么构成了开放阅读框架（ORF）和蛋白质编码RNA的假设， 生物过程的大量新调节剂的限制性鉴定。我们称之为非特征化 物质基因组暗物质在这个建议中，我们的目标是系统地识别和功能揭示其真正的 促进炎症反应。通过利用稳态和活化的核糖体分析， 在巨噬细胞中，我们已经鉴定了经历稳健翻译的“非编码”RNA。此外，我们发现 在经典注释的蛋白质编码基因内的多个ORF的广泛多顺反子翻译。到 揭示了这些替代ORF的功能贡献，并从其基因的功能中描绘出它们的作用。 注释ORF，我们建议进行一个平行的功能逆筛选方法的损失和增益，以确定 新的蛋白质，有助于炎症反应。此外，虽然蛋白质编码基因是 我们认为它们的功能仅仅是为蛋白质合成提供信息， 在细菌刺激后高度差异表达但不翻译。这是一种 质疑了蛋白质编码基因的分类。通过结合转录沉默和ORF 在破坏研究中，我们的目标是将基因RNA的功能贡献与其编码潜力脱钩。在 此外，尽管目前的核糖体分析技术不适合发现未注释的转录本， 在翻译过程中，我们已经鉴定出大量的mRNA来源于非典型的“反式剪接”， 两种不同的转录本，似乎编码新的嵌合蛋白质。通过开发一种创新的技术 管道，RiboFusionSeq，我们将产生第一个严格的识别平台编码嵌合RNA。 此外，为了揭示通过“反向剪接”的转录内环化以编码新蛋白质的能力， 将建立RiboCircSeq。使用特异性靶向反式和反向剪接的敲除方法 事件，我们将进行第一次功能筛选的蛋白质来源于非典型剪接和询问 对免疫力的贡献。最后，我们将生成动物模型来测试机械和生理 我们的发现在炎症和疾病中的重要性。总之，这些研究将提供一个变革性的 在免疫应答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， 哺乳动物基因组功能注释，3）鉴定了大量新分子以供进一步研究 和4）对理解所有人类疾病的潜在过程具有深远的影响。