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. 2020 Jan 17;15(1):132-139.
doi: 10.1021/acschembio.9b00655. Epub 2019 Dec 12.

YTHDF2 Recognition of N1-Methyladenosine (m1A)-Modified RNA Is Associated with Transcript Destabilization

Kyung W Seo  1 , Ralph E Kleiner  1
Affiliations

YTHDF2 Recognition of N1-Methyladenosine (m1A)-Modified RNA Is Associated with Transcript Destabilization

Kyung W Seo et al. ACS Chem Biol. .

Abstract

Epitranscriptomic modifications play an important role in RNA function and can impact gene expression. Here, we apply a chemical proteomics approach to investigate readers of N1-methyladenosine (m1A), a poorly characterized modification on mammalian mRNA. We find that YTHDF proteins, known m6A readers, recognize m1A-modified sequences in a methylation-specific manner. We characterize binding of recombinant YTHDF1/2 proteins to m1A-modified oligonucleotides to demonstrate that these interactions can exhibit comparable affinity to m6A-recognition events and occur in diverse sequence contexts. Further, we demonstrate YTHDF2 interacts specifically with endogenously modified m1A transcripts. Finally, we deplete cellular YTHDF2 to show that the abundance of m1A-modified transcripts is increased in its absence. Similarly, increasing m1A levels through depletion of ALKBH3, an m1A eraser protein, destabilizes known m1A-containing RNAs. Our results shed light on the function of m1A on mRNA and provide a mechanistic framework to further evaluate the role of m1A in biological processes.

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Figures

Figure 1.
Figure 1.
Chemoproteomic profiling of the m1A interactome. a) Photocrosslinking RNA probes used in this work. b) Schematic for comparative proteomics workflow. c) Volcano plot of protein enrichment from m1A proteomics experiments.
Figure 2.
Figure 2.
Characterization of YTHDF1/2-m1A binding. a) EMSA for YTHDF1 and probe 3/4. b) Quantification of YTHDF1-probe 3/4 binding. c) EMSA for YTHDF2 and probes 3 /4. d) Quantification of YTHDF2-probe 3/4 binding. e) EMSA images YTHDF1 and probes 5/6. f) Quantification of YTHDF1-probe 5/6 binding. g) EMSA for YTHDF2 and probes 5/6. h) Quantification of YTHDF2-probe 5/6 binding.
Figure 3.
Figure 3.
Characterization of YTHDC1-m1A/m6A binding. a) EMSA for YTHDC1 and probes 3, 4, or 7. b) Quantification of YTHDC1-probe 3/4/7 binding. c) EMSA for YTHDC1 and probes 5, 6, or 8. d) Quantification of YTHDC1-probe 5/6/8 binding.
Figure 4.
Figure 4.
YTHDF2-m1A regulation in living cells. a) Model for YTHDF2-m1A-mediated regulation of transcript stability. b) Venn diagram to classify transcripts as m1A-exclusive, m6A-exclusive, and both m1A- and m6A-modified. c) LC-MS/MS quantification of m1A levels in RNA enriched by immunoprecipitation of 3xFLAG-tagged YTHDF2 or G3BP1. d) Relative abundance of exclusive m1A-modified transcripts upon YTHDF2 knockdown. e) Cumulative distribution of transcript abundance upon ALKBH3 knockdown (*, p < 0.05; **, p < 0.005; ***, p < 0.0005).
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