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N6-methyladenosine-dependent regulation of messenger RNA stability. ​

Literature Information ​

DOI10.1038/nature12730
PMID24284625
JournalNature
Impact Factor48.5
JCR QuartileQ1
Publication Year2014
Times Cited2446
KeywordsN6-methyladenosine, mRNA stability, YTHDF2 protein
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0028-0836
Pages117-20
Issue505(7481)
AuthorsXiao Wang, Zhike Lu, Adrian Gomez, Gary C Hon, Yanan Yue, Dali Han, Ye Fu, Marc Parisien, Qing Dai, Guifang Jia, Bing Ren, Tao Pan, Chuan He

TL;DR ​

This study reveals that the m(6)A modification in mRNA is recognized by the YTHDF2 protein, which regulates mRNA degradation by directing mRNA from the translatable pool to decay sites, thus influencing RNA metabolism. Identifying over 3,000 RNA targets underscores the critical role of m(6)A and YTHDF2 in cellular functions and disease, highlighting their significance in mRNA regulation.

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N6-methyladenosine · mRNA stability · YTHDF2 protein

Abstract ​

N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

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Primary Questions Addressed ​

  1. What are the potential implications of m(6)A modification on gene expression regulation in various cellular contexts?
  2. How do the identified m(6)A demethylases influence the stability and degradation of mRNAs in response to environmental changes?
  3. In what ways might the YTHDF2 protein interact with other RNA-binding proteins to modulate mRNA fate?
  4. Can the understanding of m(6)A modification lead to novel therapeutic strategies for diseases associated with RNA metabolism?
  5. What experimental approaches could be employed to further elucidate the mechanisms by which m(6)A modifications affect RNA localization and degradation?

Key Findings ​

Research Background and Purpose ​

N6-methyladenosine (m6A) is the most prevalent internal modification found in messenger RNA (mRNA) of higher eukaryotes. It plays a crucial role in cell viability and development, yet its precise function remains unclear. Recent discoveries of m6A demethylases have highlighted its significance in biological processes and diseases. This study aims to elucidate the role of m6A in mRNA stability regulation through the m6A-binding protein YTHDF2.

Main Methods/Materials/Experimental Design ​

The research employs several advanced techniques to investigate the role of YTHDF2 in mRNA metabolism and stability:

  1. RNA Binding and Identification:

    • PAR-CLIP: To identify RNA targets of YTHDF2.
    • RIP-seq: To profile YTHDF2-RNA complexes.
    • m6A-seq: To analyze m6A modification levels in mRNA.

  2. Functional Analysis:

    • Ribosome Profiling: To assess the translation status of mRNAs.
    • RNA Lifetime Profiling: To measure the stability of mRNA targets post-transcription inhibition.

  3. Cell Culture and Transfection: HeLa cells were used for experiments involving siRNA knockdown of YTHDF2 and MT-A70 (the m6A methyltransferase).

  4. Protein Interaction Studies: The localization and interaction of YTHDF2 with mRNAs were assessed using fluorescence microscopy and co-immunoprecipitation.

  5. Data Analysis: The sequencing data were analyzed for RNA expression levels, binding affinities, and statistical significance using various bioinformatics tools.

Mermaid diagram

Key Results and Findings ​

  • YTHDF2 Interaction: YTHDF2 selectively binds to m6A-containing mRNAs, with a strong preference for the m6A consensus motif. Over 3,000 RNA targets were identified, predominantly mRNAs.
  • RNA Decay Mechanism: YTHDF2 promotes the degradation of its target mRNAs by localizing them to processing bodies (P-bodies), thus affecting their translation and stability.
  • Stability Changes: Knockdown of YTHDF2 resulted in increased stability of mRNA targets (average lifetime increased by ~30%) and altered their distribution between translatable and non-translatable pools.
  • Translation Efficiency: A significant reduction in translation efficiency was observed for YTHDF2 targets upon its knockdown, suggesting its primary role in mRNA degradation rather than translation initiation.

Main Conclusions/Significance/Innovation ​

The study presents the first functional demonstration of a m6A reader protein, YTHDF2, and its role in mRNA metabolism. It establishes a model where m6A modifications dynamically regulate mRNA stability and localization through selective binding by YTHDF2, thereby influencing gene expression and cellular processes.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focuses on HeLa cells, which may not fully represent all cell types. Additionally, the long-term effects of YTHDF2 knockdown on cellular viability were not extensively explored.
  • Future Directions: Further research could investigate the role of YTHDF2 in different cell types and its implications in various diseases. Additionally, exploring other m6A reader proteins could provide a broader understanding of m6A's role in RNA biology.

Summary Table ​

SectionKey Points
Research Backgroundm6A modification is crucial for mRNA stability and cellular functions.
MethodsPAR-CLIP, RIP-seq, m6A-seq, ribosome profiling, RNA lifetime profiling, fluorescence microscopy.
Key ResultsYTHDF2 binds m6A-containing mRNAs, promoting their decay and influencing translation efficiency.
ConclusionsYTHDF2 is a key m6A reader that regulates mRNA stability and localization.
LimitationsFocus on HeLa cells; long-term effects not fully explored.
Future DirectionsInvestigate YTHDF2 in various cell types and diseases; explore other m6A reader proteins.

References ​

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Literatures Citing This Work ​

  1. RNA: The (methylation) reader. - Kim Baumann - Nature reviews. Molecular cell biology (2014)
  2. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. - Xiao-Li Ping;Bao-Fa Sun;Lu Wang;Wen Xiao;Xin Yang;Wen-Jia Wang;Samir Adhikari;Yue Shi;Ying Lv;Yu-Sheng Chen;Xu Zhao;Ang Li;Ying Yang;Ujwal Dahal;Xiao-Min Lou;Xi Liu;Jun Huang;Wei-Ping Yuan;Xiao-Fan Zhu;Tao Cheng;Yong-Liang Zhao;Xinquan Wang;Jannie M Rendtlew Danielsen;Feng Liu;Yun-Gui Yang - Cell research (2014)
  3. Methyltransferases modulate RNA stability in embryonic stem cells. - Shuibin Lin;Richard I Gregory - Nature cell biology (2014)
  4. Nucleic acid oxidation in DNA damage repair and epigenetics. - Guanqun Zheng;Ye Fu;Chuan He - Chemical reviews (2014)
  5. Gene expression regulation mediated through reversible m⁶A RNA methylation. - Ye Fu;Dan Dominissini;Gideon Rechavi;Chuan He - Nature reviews. Genetics (2014)
  6. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. - Kate D Meyer;Samie R Jaffrey - Nature reviews. Molecular cell biology (2014)
  7. Genome-wide mapping of cellular protein-RNA interactions enabled by chemical crosslinking. - Xiaoyu Li;Jinghui Song;Chengqi Yi - Genomics, proteomics & bioinformatics (2014)
  8. RNA epigenetics. - Nian Liu;Tao Pan - Translational research : the journal of laboratory and clinical medicine (2015)
  9. Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation. - Chao Xu;Ke Liu;Wolfram Tempel;Marina Demetriades;WeiShen Aik;Christopher J Schofield;Jinrong Min - The Journal of biological chemistry (2014)
  10. Reading RNA methylation codes through methyl-specific binding proteins. - Xiao Wang;Chuan He - RNA biology (2014)

... (2436 more literatures)

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