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Structural basis of YTH domain as m6A reader

ABSTRACT: The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.

ORGANISM(S): Homo sapiens

SUBMITTER: Chuan He

PROVIDER: E-GEOD-58352 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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m6A-dependent regulation of messenger RNA stability

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNA of all higher eukaryotes. Here we present that m6A is selectively recognized by human YTH domain family (YTHDF2) protein to regulate mRNA degradation. By using crosslinking and immunoprecipitation, we have identified over 4000 substrate RNA of YTHDF2 with conserved core motif of G(m6A)C. We further estabilshed the role of YTHDF2 in RNA metabolism by a combination of ribosome profiling, RNA sequencing, m6A level quantification and cell-based imaging: the C-terminal domain of YTHDF2 selectively binds to m6A of mRNA and the N-terminal domain is responsive for localizing mRNA from translatable pool to processing body where mRNA decay occurs. PAR-CLIP and RIP was used to identify YTHDF2 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF2 siRNA knock-down

2013-11-29 | E-GEOD-49339 | biostudies-arrayexpress

N6-methyladenosine Modulates Messenger RNA Translation Efficiency

Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down

2015-06-09 | E-GEOD-63591 | biostudies-arrayexpress

Structural basis of YTH domain as m6A reader

Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader.

2014-09-18 | GSE58352 | GEO

Homo sapiens

Project description:Structural basis of YTH domain as m6A reader

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MS-based characterization of YTHDC1 interactome

Project description:Heat shock induces a cell response leading to profound changes in genome expression. Recently, N6-methyladenosine (m6A) RNA modification has been implicated in this response, but with limited information of its role in the heat-induced reprograming of gene expression. Most of m6A molecular and cellular functions rely on m6A readers and the best characterized m6A readers are members of the YTH-domain-containing protein family present from yeast to humans. To investigate the function of the nuclear m6A reader YTHDC1, we characterized its binding partners.

2023-01-11 | PXD037414 | Pride

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition

Project description:The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by ‘reader’ proteins like those of the YTH family that recruit additional factors to alter splicing, RNA stability or translation. Germline-specific YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here we use a tagged mouse line to identify U-rich motifs as binding sites of YTHDC2 on 3′ UTRs of testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mouse lacking this ability displays no obvious phenotypes. Its 3′→5′ RNA helicase activity is essential for fertility, with the catalytic-dead mutation being dominant-negative. Single-cell transcriptomics reveal that Ythdc2 mutant mitotic testicular germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity. We also reveal a conserved role for YTHDC2 in animal fertility.

2022-03-22 | GSE196427 | GEO

Interactome of Toxoplasma gondii Pre-mRNA 3’ Processing Complexes

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2021-07-16 | PXD024326 | Pride

Recognition of m6A RNA by the non-canonical reader IMP1

Project description:m6A methylation is the prevalent post-transcriptional modification in eukaryotic mRNAs and provides an essential layer of regulation in organismal development and in disease. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by the binding of an expanding list of m6A readers. An important question is how protein readers that do not contain a canonical m6A-specific YTH domain recognize methylated RNA. Here, we show that the non-canonical reader IMP1 directly recognises the m6A group using a dedicated hydrophobic platform in the KH4 domain, creating a stable and high affinity interaction with the methylated RNA targets. Notably, the recognition of the m6A group is independent from the underlying sequence context, but is layered upon IMP1 strong sequence specificity for GGAC RNA. Together, our data indicate that, contrarily to the well-characterised YTH readers, IMP1 recognises and binds both m6A-methylated and non-methylated RNA targets with high affinity. This suggests that m6A methylation does not provide a general layer of control of IMP1 function, but rather plays a directed role in specific regulatory pathways.

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Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and Î±-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates

2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress

Regulation of m6A transcripts by the 3ʹ→5ʹ RNA helicase YTHDC2 is essential for a successful meiotic program in the mammalian germline

Project description:N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. In mutant males, mitotic spermatogonia fail to differentiate into meiotic spermatocytes. Analysis identifies transcripts upregulated in the Yhtdc2 mutant testes to be those that are expressed in purified spermatogonia, while high-throughput mapping of the m6A transcriptome in the mouse male germline demonstrates the upregulated transcripts to be those that are enriched in m6A marks. Our biochemical studies indicate that YTHDC2 is an RNAinduced ATPase that fuels its 3ʹ→5ʹ RNA helicase activity. Interestingly, we find an RNA-independent interaction between the 5ʹ→3ʹ exoribonuclease XRN1 and the Ankyrin repeat of YTHDC2 that is wedged in between the two RecA helicase domains. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts that is essential for successful meiosis.

2017-10-14 | GSE102346 | GEO

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