Identification of YTH Domain-Containing Proteins as the Readers for N1-Methyladenosine in RNA

Abstract

N1-methyladenosine (m¹A) is an important post-transcriptional modification in RNA; however, the exact biological role of m¹A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m¹A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m¹A in RNA. We also found that Trp⁴³² in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N⁶-methyladenosine (m⁶A), is required for its recognition of m¹A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m¹A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m¹A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m¹A in RNA and provided new insight into the functions of m¹A in RNA biology.

Figure 1.

Identification of m¹A-binding proteins. (a) Schematic overview of the SILAC-based quantitative proteomics method for discovering m¹A-binding proteins. Shown is the workflow for a forward SILAC labeling experiment. (b,c) Scatterplot of the proteins identified from pull-down assays using m¹A- (b) or m⁶A- (c) carrying RNA over the corresponding unmodified RNA and the whole-cell protein lysate of HEK293T cells. The proteins present in top right quadrant are m¹A- (b) or m⁶A (c)-binding proteins, whereas those in the bottom left quadrant are proteins which bind more weakly to the m¹A- or m⁶A-bearing RNA than the control RNA probe. The proteins that fall in the bottom right quadrant represent those proteins displaying large light/heavy ratios in both forward and reverse SILAC labeling experiments, which are likely due to incomplete SILAC labeling (for those proteins with slow turnover rate). The data were based on results from two forward and one reverse SILAC experiments.

Figure 2.

Representative ESI-MS for the [M + 2H]²⁺ ions of a tryptic peptide of YTHDF2, SINNYNPK (a, b) and a tryptic peptide of YTHDF3, IGGDLTAAVTK (c, d) revealing the preferential binding of YTHDF2 and YTHDF3 toward the m¹A-containing RNA probe in both forward (a, c) and reverse (b, d) SILAC experiments.

Figure 3.

YTH domain-containing proteins bind directly to m¹A-carrying RNA. (a−d) Electrophoretic mobility shift assay (EMSA) for measuring the binding affinity of YTHDF1 (a), YTHDF2 and W432A mutant proteins (b), YTHDF3 (c), and YTH domain of YTHDC1 (d) with m⁶A, A, and m¹A-carrying RNA probes. The K_d values (in μM) are listed in (a)−(d), and error bar represents the SEM (n = 3). (e) Overlap between the m¹A sites (200 bp window centered on the m¹A sites) and YTHDF1–3, and YTHDC1 PAR-CLIP binding sites in HeLa cells.