N6-methyl-adenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA) and has been implicated in many cellular processes, including splicing, mRNA stability, translational activity, and cytoplasmic localization. Moreover, deficits in m6A methylation are linked to human diseases, such as obesity, developmental deficits, cancer, and neurodegenerative disorders. Studies in our laboratory and others indicate that m6A can influence cellular processes by directly recruiting ?reader? proteins or by regulating the accessibility of RNA-binding proteins through alteration of local RNA structure (called the m6A-switch). However, despite the rapidly growing interest in and importance of m6A, the methods available for mapping m6A have considerable drawbacks, including the requirement of large amounts of sample input and the inability to determine the modification stoichiometry, a known variable of the m6A epitranscriptome. The proposed research aims to overcome these limitations by developing a novel deaminase-based m6A-seq method (Deam-seq) that will allow for determination of the m6A modification fraction at single-base resolution. Moreover, since this approach requires no enrichment step and is enzyme-based, it should be readily applicable for characterization of biologically limited samples, such as clinical specimens. If successful, we anticipate that this new technology will be extremely powerful in monitoring epitranscriptional dynamics and characterizing unique cell types within a population. Moreover, I will also test the hypothesis that the methylation fraction plays an important role in regulating mRNA splicing. Finally, given the emerging evidence that intronic m6A residues may be far more common than suggested based on conventional m6A-seq approaches, I will also develop new methodology for global determination of the m6A landscape within introns and nuclear circular RNAs. Overall, this research should yield many new insights into how m6A modulates mRNA fate.
N6-methyl-adenosine (m6A) is a prevalent modification in messenger and long non-coding RNAs that plays important roles in regulating gene expression. m6A methylation has been implicated in numerous processes, including differentiation, cancer progression, and brain development. The proposed research will establish new high-throughput sequencing methods that determine the m6A modification fraction at single-base resolution (Deam-seq) and provide the first in-depth profile of m6A in intronic regions and nuclear circular RNAs (Circtron- m6A-seq).
