Uncovering the regulatory principles of dynamic mRNA methylation

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Project Summary Chemical modification of RNA is a critical mechanism of gene expression regulation, controlling RNA processing, stability, and, in the case of mRNA, translation. The chemical diversity of RNA modifications suggests that there is extensive biology yet to be uncovered. A recent surge in this field has been driven largely by rapid advances in high throughput sequencing methods that allow us to map these marks on a transcriptome-wide scale. However, the majority of studies remain correlative and are not able to reveal the molecular mechanisms of RNA modification function or regulation. The information we glean from such studies often represents an average across all transcripts in the cell, and does not take into account the spatial organization or temporal control of RNA transcription, processing, and trafficking. Moreover, the majority of work on mRNA modifications has focused on a single modification, N6-methyladenosine (m6A), which represents only one of over one hundred modifications annotated to date. Here we describe two research directions in our laboratory that address fundamental questions about mRNA modifications. The first research direction aims to characterize a new mRNA modification, N1-methyladenosine (m1A). Since discovering this modification in mRNA in during my postdoctoral work, technical challenges have prevented us from understanding the functions and regulation of this mRNA modification. These challenges have also led to conflicting data and controversy over the presence and prevalence of m1A in mRNA. We have recently validated new m1A mRNA sites and we are now well positioned to finally shed light on this mysterious mRNA modification. The second research direction aims to identify the mechanisms that coordinate cotranscriptional modification of mRNAs as they are being synthesized. This remains mysterious for even the most well- characterized mRNA modification, m6A. We hypothesize that RNA polymerase II complexes recruit RNA modification enzymes during transcription, akin to how splicing and processing factors are cotranscriptionally recruited to nascent mRNA. Using detailed biochemistry and RNA labeling approaches, we will dissect the mechanisms of cotranscriptional RNA methylation. To tackle challenges such as these, our lab identifies specific settings, essentially molecular model systems, that allow us to dissect the molecular mechanisms of RNA modification-mediated regulation of gene expression. Once we reveal the regulatory components and principles that govern these specific systems, we then have a molecular foothold to determine how general or specific these principles are, and in which contexts they apply. Our work will open up a new areas of RNA biology and allow us to understand how defects in these mechanisms result in associated human diseases.