Abstract
N6A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N6-methylated adenosine reader domain and report its solution structure in complex with a N6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m6A recognition. These findings establish a molecular function for YTH domains as m6A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m6A recognition.
Highlights
Methylation of adenine at the N6 position (m6A) is considered the most abundant messenger RNA modification in eukaryotes besides the 5 cap structure [1,2]
The sequence determined by systematic evolution of ligands by exponential enrichment (SELEX) for YT521-B allowed us to map the RNA-binding surface of the YT521-B homology (YTH) domain, the fact that many protein resonances were absent in the spectra of the complex prevented us to determine its structure [17]
Nuclear magnetic resonance (NMR) spectra of the YTH domain of YT521 bound to 5 -UGACAC-3 displayed a larger number of peaks with sharper resonance linewidth as compared to the SELEXderived sequence
Summary
Methylation of adenine at the N6 position (m6A) is considered the most abundant messenger RNA modification in eukaryotes besides the 5 cap structure [1,2]. Two out of three top confidence category proteins enriched in the pull-downs with the methylated RNA from HepG2 cell lysates contained one YTH domain (YTHDF2, YTHDF3) [7]. Full-length proteins YTHDF1, which contains a YTH domain, YTHDF2 and YTHDF3 were later shown by gel shifts to have increased affinities for the methylated compared to the non-methylated form of the same RNA target sequence [12]. This suggested that YTH-containing proteins, whose functions are generally unknown, could act as m6A readers
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