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

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。尽管有大量的 加速和广泛使用转录切割方法来理解生物过程，我们仍然在这样做 不能从根本上理解RNA翻译的最基本层面。传统的定义，将 作为蛋白质编码或非编码的RNA，目前与全基因组的最新发现不相容 翻译学研究。我们假设，技术壁垒和对教条的坚持相结合 关于开放阅读框架(ORF)和编码RNA的蛋白质的假设严重 生物过程中过多的新调节器的受限识别。我们称这为无特征的 物质基因组暗物质。在这项提案中，我们的目标是系统地识别并从功能上揭示其真实情况 对炎症反应的贡献。通过利用稳定状态和激活的核糖体图谱 巨噬细胞，我们已经鉴定出“非编码”的RNA正在进行强健的翻译。此外，我们还发现了 经典注释的蛋白质编码基因中多个开放阅读框的广泛多顺反子翻译。至 揭示这些替代ORF的功能贡献，并将它们的作用与它们的基因的作用区分开来 我们提出了一种并行的损失和增益函数逆筛选方法来识别 参与炎症反应的新蛋白质。此外，尽管蛋白质编码基因是 被认为仅通过为蛋白质合成提供信息来发挥作用，我们已经确定了一类mRNAs 在细菌刺激下高度差异表达但不翻译的基因。这在哲学上是 对蛋白质编码基因的分类提出质疑。通过结合转录沉默和开放阅读框架 在干扰研究中，我们的目标是将基因RNA的功能贡献与其编码潜力脱钩。在……里面 此外，尽管目前的核糖体图谱技术不适合发现未注释的转录本 在翻译过程中，我们发现了过多的mrna来自于非典型的 两种不同的转录本似乎编码了新的嵌合蛋白。通过开发创新的技术 管道，RiboFusionSeq，我们将为编码嵌合RNA生成第一个严格的鉴定平台。 此外，为了揭示通过“后剪接”来编码新蛋白质的转录内循环的能力， 将建立RiboCircSeq。使用专门针对反式剪接和反向剪接的击倒方法 事件，我们将对来自非典型剪接的蛋白质进行第一次功能筛选和询问 他们对豁免权的贡献。最后，我们将生成动物模型来测试机制和生理 我们的发现在炎症和疾病方面的重要性。总而言之，这些研究将提供一种变革性的 在免疫应答过程中蛋白质编码基因组的分辨率水平，2)建立一个新的范例 哺乳动物基因组的功能注释，3)识别大量新的分子以供进一步研究 以及4)对理解所有人类疾病背后的过程具有深远的影响。

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.