Ping Yin 
Publications
AI-empowered integrative structural characterization of m<sup>6</sup>A methyltransferase complex.
Yan Xuhui X Pei Kai K Guan Zeyuan Z Liu Feiqing F Yan Junjun J Jin Xiaohuan X Wang Qiang Q Hou Mengjun M Tang Chun C Yin Ping P
Cell research 20221110 12
Similar Datasets
Project description:N6-methyladenosine (m6A) is the most abundant ribonucleotide modification among the eukaryotic messenger RNAs (mRNAs). The m6A “writer” is composed of an m6A-METTL complex (MAC), the catalytic subunit, and an m6A-METTL associated complex (MACOM), the regulatory subunit essential for the enzymatic activity. Here we report the cryo-electron microscopy (cryo-EM) structures of MACOM at 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and ZC3H13 stretches the conformation by binding VIRMA. The lower 4.4-Å resolution cryo-EM datamap of MACOM in complex with MAC, in combination with crosslinking mass spectrometry and GST-pulldown analysis, elucidates a plausible model of the m6A writer complex, in which MACOM binds MAC mainly through WTAP and METTL3 interaction and both components directly contact with RNA substrates revealed by 4-thiouridine-labeled RNA crosslinking analysis. This work establishes the possible assembly mechanism of MACOM and MAC as an active m6A writer for RNA substrate recognition and modification.
2022-06-23 | PXD028456 | iProX
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. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
2014-09-18 | E-GEOD-58352 | biostudies-arrayexpress
Project description:The methyltransferase complex (m6A writer), which catalyzes the deposition of N6-methyladenosine (m6A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, were discovered as novel components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA. Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation. In addition, HIZ1 was found to be involved in root hair development upon auxin transport inhibition in a HAKAI-dependent manner. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ1 appears to be a HAKAI-dependent negative regulator of m6A deposition and HIZ2 is a novel and essential member of the Arabidopsis m6A writer complex.
2022-02-09 | PXD026287 | Pride
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
Project description:N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerisation and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds new functional and molecular insights into the mechanism of the m6A mRNA writer complex.
2021-04-29 | PXD022294 | Pride
Project description:In order to obtain a stringent m6A methylome in Drosophila adults, we performed m6A-RIP-seq in yw control (male and female), Mettl3 (male), Mettl14 (male) and Hakai (male) mutant flies. We find that the effective m6A modification, which depends on the writer complex, is mostly distributed in 5’ UTR and near start codon in Drosophila, in contrast to the mammalian system. We define a set of high-confident m6A methylation sites shared by Mettl3, Mettl14 and Hakai, indicating that Hakai is a core component of the m6A writer complex. We also find differential methylation pattern in certain loci between male and female flies.
2021-03-01 | GSE155662 | GEO
Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates
2016-06-24 | E-GEOD-66086 | biostudies-arrayexpress
Project description:Using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, were discovered as novel components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA. Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation. In addition, HIZ1 was found to be involved in root hair development upon auxin transport inhibition in a HAKAI-dependent manner. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ1 appears to be a HAKAI-dependent negative regulator of m6A deposition and HIZ2 is a novel and essential member of the Arabidopsis m6A writer complex.
2022-01-21 | GSE188610 | GEO
Project description:Using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, were discovered as novel components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA. Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation. In addition, HIZ1 was found to be involved in root hair development upon auxin transport inhibition in a HAKAI-dependent manner. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ1 appears to be a HAKAI-dependent negative regulator of m6A deposition and HIZ2 is a novel and essential member of the Arabidopsis m6A writer complex.
2022-01-20 | GSE174573 | GEO
Project description:In this study we identify Mettl3, an m6A RNA modification writer, as a critical regulator for terminating naM-CM-/ve pluripotency and a positive maintainer of primed pluripotency in vitro and in vivo. Remarkably, Mettl3 knockout pre-implantation epiblasts and naM-CM-/ve ES cells, entirely lack m6A on coding mRNAs and are viable. Yet, they fail to adequately terminate the naM-CM-/ve pluripotent state, and subsequently undergo aberrant priming and early lineage commitment at the post-implantation stage. A comprehensive functional and genomic analysis involving profiling of m6A, RNA transcription and translation in Mettl3 wild-type and knockout pluripotent and differentiated cells, identified m6A as a critical determinant that destabilizes secondary naM-CM-/ve specific pluripotency genes Esrrb, Klf4 and Nanog, and restrains their transcript stability and translation efficiency. In summary, our findings provide for the first time evidence for a critical role for an mRNA epigenetic modification in early mammalian development in vivo, and identify a mechanism that functionally regulates mouse naM-CM-/ve and primed pluripotency in an opposing manner. m6A-seq was measured from total RNA in mouse embryonic stem cells (ESCs), embroid bodies (EBs) and embronic fibroblasts (MEF). 3 biological replicates are available from BVSC ESC line and EBs, and two biological replicates are available for MEFs. Each sample consist of IP to m6A and control input
2014-11-02 | E-GEOD-61995 | biostudies-arrayexpress