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.