RNA functions as a central hub between DNA and protein, and understanding its regulation is critical to illuminating gene expression programs in normal and disease physiology. The properties of messenger RNA (mRNA) can be modulated by dynamic chemical modifications (the epitranscriptome) that occur post- transcriptionally, such as N6-methyladenosine (m6A), which regulates mRNA turnover, translation, nuclear export, and splicing, as well as other modifications on the nucleobases. A major challenge is to identify the functional consequences of these modifications and elucidate the molecular mechanisms by which they control mRNA biology. This proposal seeks to fill this knowledge gap by developing and applying chemical biology strategies to characterize proteins that mediate the effects of mRNA modifications on cellular processes. The epitranscriptome is shaped by RNA-modifying enzymes (writers and erasers) and interpreted by modification-specific RNA-binding proteins (readers). Characterizing these protein-RNA interactions is critical for understanding the function and regulation of specific modifications. Previously, we developed and applied a chemical proteomics strategy to profile binders of m6A. We identified new m6A readers as well as proteins that bind preferentially to unmodified RNA. Herein, we will interrogate the role of these m6A-mediated protein-RNA interactions on mRNA behavior in the cell and further develop our approach to profile readers of another methylation mark, N1-methyladenosine (m1A). Additionally, we propose novel methodologies to characterize modified RNA-protein interactions in vitro and in the cell. Our project has the following specific aims:
Aim 1. Identify and functionally interrogate proteins that read mRNA methylation marks. We will focus on identifying and studying readers of m6A and m1A.
Aim 2. Profile the substrate specificity of mRNA methylation readers and erasers by in vitro selection.
Aim 3. Investigate the trafficking of methylated mRNA to cellular stress granules using an RNA proximity ligation strategy. Our findings will reveal how mRNA methylation regulates protein-RNA interactions to control gene expression. These studies should improve our understanding of fundamental RNA regulatory mechanisms and provide powerful and general strategies for interrogating the function of mRNA modifications.

Public Health Relevance

Normal cell physiology relies on the accurate and regulated transfer of information from DNA to RNA to protein, and errors in this process result in alterations to gene expression that can lead to disease. This proposal seeks to investigate the mechanisms by which chemical modifications on RNA control gene expression. Since proteins regulating RNA modification levels have been associated with disease, our findings will not only provide a molecular framework to understand the function of RNA modifications, but also shed light on mechanisms involved in cellular dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM132189-02S1
Application #
10134578
Study Section
Program Officer
Fabian, Miles
Project Start
2019-04-01
Project End
2024-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Princeton University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08543