Viral modulation of epitranscriptomic mechanisms

项目摘要 该提案的总体目标是开发应用于生物样品的高通量质谱技术。 研究转录后RNA修饰（PTrM）和与RNA结合蛋白（RBP）的关系。 有效的RNA加工和蛋白质翻译需要与许多RNA结合蛋白相互作用， 可以通过化学RNA修饰来调节。PTrM涉及多种过程， RNA剪接、核输出、稳定性和翻译。PTrMs控制RNA命运的机制 开辟了一个新的领域，被称为“外延脚本学”。虽然有抗体方法来检测一些 修改，需要正交方法来无偏地识别和分析PTrM。以来 在细胞核中复制的小DNA病毒必须利用细胞机制来转录和翻译 它们的基因产物，并开发出对抗宿主防御的方法，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，以破译RNA 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 产生病毒后代。根据我们合作团队生成的初步数据， 本提案的目的是采用腺病毒作为模型系统，研究非编码和 信使病毒RNA以及它们如何被利用来对抗宿主防御并促进有效的病毒RNA 加工和后代生产。腺病毒是大型非进化病毒，包括50多种不同的腺病毒。 在人类中引起广泛影响的菌株，从呼吸道感染到危及生命的器官 免疫系统较弱的人的问题。腺病毒劫持宿主细胞机器表达 病毒基因，这是通过超越RNA介导的过程来实现的。我们的初步数据显示 腺病毒感染利用RNA上的m6 A修饰来促进剪接并改变RNA蛋白 交互.在这里，我们将开发改进的质谱（MS）技术，以定量和 在感染的详细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，以确定如何利用修改 通过腺病毒来促进生长和对抗宿主防御，最终导致人类疾病。这些 高通量无偏MS技术和方法将广泛适用于实现新的 病毒-宿主相互作用和表观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.