Viral modulation of epitranscriptomic mechanisms

项目总结 这项提案的总体目标是开发高通量质谱仪技术，应用于 转录后RNA修饰(PTrm)及其与RNA结合蛋白(RBPs)关系的研究。 有效的RNA处理和蛋白质翻译需要与大量的RNA结合蛋白相互作用，并且 可以通过化学RNA修饰来调节。PTrM参与了各种不同的过程，如 RNA剪接、核输出、稳定性和翻译。PTrms控制RNA命运的机制已经 开辟了一个名为“墓志译本”的新领域。尽管有抗体方法可以检测到一些 为了改进，需要采用正交方法来无偏地识别和分析PTrM。自.以来 在细胞核中复制的小型DNA病毒既要使用细胞机械来转录和翻译 它们的基因产物，并开发出抵消宿主防御的方法，这些病毒利用和操纵 细胞RNA加工途径。因此，病毒感染提供了优雅的生物学模型来破译RNA是如何 转录及其化学修饰可以被调节和利用来指导宿主细胞机制。 致力于生产病毒后代。根据我们的协作团队生成的初步数据，我们的 本方案的目的是使用腺病毒作为模型系统来研究非编码和 信使病毒RNA及其如何被利用来对抗宿主防御和促进有效的病毒RNA 加工和后代生产。腺病毒是大的非发育病毒，包括50多种不同的 在人类身上引起广泛影响的菌株，从呼吸道感染到危及生命的器官 免疫系统减弱的人的问题。腺病毒劫持宿主细胞机表达 病毒基因，这是通过超越RNA介导的过程实现的。我们的初步数据显示 腺病毒感染利用RNA上的m6A修饰促进剪接，并改变RNA-蛋白质 互动。在这里，我们将开发改进的质谱学(MS)技术，以定量和 在详细的感染时间过程中，全面检测RNA修饰和RNA-蛋白质相互作用。 我们的MS技术将与细胞和遗传分析配对，以确定如何利用修饰 通过腺病毒促进生长和对抗宿主防御，最终导致人类疾病。这些 基于MS的高吞吐量、无偏见的技术和方法将广泛适用于实现 病毒-宿主相互作用和表位转录组学中的生物学。

PROJECT SUMMARY The overall goal of this proposal is to develop high-throughput mass spectrometry technologies applied to the study of post-transcriptional RNA modifications (PTrMs) and associations with RNA binding proteins (RBPs). Efficient RNA processing and protein translation requires interactions with numerous RNA binding proteins, and can be regulated by chemical RNA modifications. PTrMs have been implicated in such diverse processes as RNA splicing, nuclear export, stability, and translation. The mechanisms by which PTrMs control RNA fate has opened up a new field dubbed “Epitranscriptomics”. Although there are antibody approaches to detect some modifications, there is a need for orthogonal approaches for unbiased identification and analysis of PTrMs. Since small DNA viruses that replicate in the nucleus have both to employ cellular machinery to transcribe and translate their gene products, and also develop ways to counteract host defenses, these viruses harness and manipulate cellular RNA processing pathways. Virus infections thus provide elegant biological models to decipher how RNA transcription and its chemical modifications can be regulated and exploited to direct the host cell machinery towards production of viral progeny. Based on preliminary data generated by our collaborative team, our objectives in this proposal are to employ Adenovirus as a model system to study PTrMs on non-coding and messenger viral RNAs and how they are exploited to counter host defenses and promote efficient viral RNA processing and progeny production. Adenoviruses are large non-eveloped viruses and include over 50 distinct strains which elicit a wide range of effects in humans, from respiratory infections to life-threatening organ problems in people with weakened immune systems. Adenoviruses hijack the host cell machinery to express viral genes, and this is achieved by overtaking RNA-mediated processes. Our preliminary data show how Adenovirus infection exploits the m6A modification on RNA to promote splicing and also alters RNA-protein interactions. Here we will develop improved mass spectrometry (MS) technologies to quantitatively and comprehensively detect RNA modifications and RNA-protein interactions over a detailed time-course of infection. Our MS technology will be paired with cellular and genetic assays to determine how the modifications are utilized by Adenovirus to promote growth and counter host defenses in ways that culminate in human disease. These high-throughput unbiased MS-based technologies and approaches will be broadly applicable to enable new biology in virus-host interactions and epitranscriptomics.