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： 尽管哺乳动物蛋白质编码基因组的注释数量庞大，但其注释却极其不完整。 加速和广泛使用转录组学方法来理解生物过程，我们仍然在做 没有从根本上理解 RNA 翻译的最基本水平的传统定义。 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相容 我们努力克服技术障碍和对教条主义的坚持。 关于什么构成开放阅读框（ORF）和蛋白质编码 RNA 的假设，严重影响了 对大量新型生物过程调节因子的有限鉴定，我们称之为“未表征的”。 在这个提案中，我们的目标是系统地识别和功能性地揭示其真实的物质。 通过利用稳态和激活的核糖体分析来促进炎症反应。 在巨噬细胞中，我们发现了正在进行稳健翻译的“非编码”RNA。 经典注释的蛋白质编码基因内多个 ORF 的广泛多顺反子翻译。 揭示这些替代 ORF 的功能贡献，并根据其基因的作用描述它们的作用 带注释的ORF，我们建议进行并行的功能损失和增益逆筛选方法来识别 此外，尽管蛋白质编码基因是促进炎症反应的新蛋白质。 被认为仅通过提供蛋白质合成信息来发挥作用，我们已经鉴定了一类 mRNA 在细菌刺激后高度差异表达，但在哲学上没有被翻译。 通过转录沉默和 ORF 质疑蛋白质编码基因的分类。 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。 此外，尽管当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源自非典型“反式剪接”的 mRNA 通过开发创新技术，两种不同的转录物似乎编码新型嵌合蛋白。 管道 RiboFusionSeq，我们将生成第一个用于编码嵌合 RNA 的严格鉴定平台。 此外，为了揭示通过“反向剪接”进行转录内环化以编码新蛋白质的能力， 将使用专门针对反式剪接和反向剪接的敲除方法来建立 RiboCircSeq。 事件发生后，我们将对源自非典型剪接的蛋白质进行首次功能筛选并询问 最后，我们将生成动物模型来测试其机制和生理学。 我们的发现在炎症和疾病方面的重要性。总的来说，这些研究将 1) 提供变革性的成果。 免疫反应期间蛋白质编码基因组的分辨率水平，2）建立新的范例 哺乳动物基因组的功能注释，3) 鉴定大量新分子以供进一步研究 4）对于理解所有人类疾病的潜在过程具有深远的影响。