Project Summary/Abstract Chemical modifications on mammalian messenger RNA (mRNA) have recently been shown to play critical and diverse regulatory roles in mRNA metabolism and translation. For example, the most abundant mRNA modification, N6-methyladenosine (m6A), is crucial for mammalian stem cell differentiation and tissue development in almost all systems tested so far. Dedicated proteins, many of which are essential in mammals, have evolved to install, recognize, and remove m6A marks (writers, readers, and erasers, respectively). Dysregulation of m6A methylation has been connected to a variety of human diseases and disorders. Functional roles have also been proposed for other internal modifications present in mammalian mRNA, including, but not limited to: pseudouridine (Ψ), 5-methylcytosine (m5C), 2’-O-methylation (Nm), N1-methyladenosine (m1A), N7- methylguanosine (m7G), and N3-methylcytosine (m3C). Our most recent research has uncovered modifications on chromosome-associated regulatory RNAs (carRNAs), such as promoter-associated RNA (paRNA), enhancer RNA (eRNA), and repeat RNAs, as well as frequent modifications in introns of pre-mRNA. The carRNA modifications have been shown to regulate chromatin state and transcription, and intron modifications may affect pre-mRNA processing. Despite rapid advances in the discovery and functional characterization of various RNA modifications and their effector proteins, a significant bottleneck limits the entire field of epitranscriptomics research: a dearth of quantitative sequencing methods that can comprehensively map most RNA modifications at base resolution with exact modification fraction information. The availability of such methods is critical for assessing the importance of these modifications in different regions of mRNA, examining the effects of dynamic changes in modification fraction, assigning modifications to different writers and analyzing their functional relevance, identifying target transcripts and sites of demethylation and analyzing their functional relevance, discovering new effectors for RNA modifications by overlapping with known RBP-binding sites or genomics features, and evaluating the physiological consequences of RNA modifications in biological processes. We have established both nucleic acid chemistry and directed protein evolution platforms to invent new technologies that transform RNA modifications to be read out as mutations or deletions that are universally compatible with extant sequencing platforms. Computational pipelines and RNA modification databases will be built to support the new method development and epitranscriptome research in the broad community. These new technologies will be optimized to work on low-input samples, particularly neuronal and clinical samples. We will focus on integrating new methods into robust protocols to map multiple RNA modifications in single experiments. Our proposed research will deliver high-throughput, high-resolution, and high-sensit...