Publications

Authors Eckwahl M, Xu RY, Michalkiewicz J, Zhang W, Patel P, Cai Z, Pan T

Abstract RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterize the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We find that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we map at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we show that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA.Importance: Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identify unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely map its location, and show that ALYREF, a “reader” protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.
Journal Name
J. Virology 2020, In Press

Links
PDF

Authors Wu T, Lyu R, You Q, He C.

Abstract Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as ‘transcription bubbles’. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.
Journal Name
Nat. Methods 2020, In Press

Links
PDF

Authors Hao Z, Wu T, Cui X, Zhu P, Tan C, Dou X, Hsu KW, Lin YT, Peng PH, Zhang LS, Gao Y, Hu L, Sun HL, Zhu A, Liu J, Wu K J, He C.

Abstract N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.
Journal Name
Mol. Cell 2020, S1097-2765: 30111-30118.

Links
PDF

Authors Shafik AM, Allen EG, Jin P

Abstract N6-methyladenosine (m6A) is a dynamic RNA modification that regulates various aspects of RNA metabolism and has been implicated in many biological processes and transitions. m6A is highly abundant in the brain; however, only recently has the role of m6A in brain development been a focus. The machinery that controls m6A is critically important for proper neurodevelopment, and the precise mechanisms by which m6A regulates these processes are starting to emerge. However, the role of m6A in neurodegenerative and neuropsychiatric diseases still requires much elucidation. This review discusses and summarizes the current body of knowledge surrounding the function of the m6A modification in regulating normal brain development, neurodegenerative diseases and outlines possible future directions.
Journal Name
Epigenomics 2020, 12: 371-380.

Links
PDF

Authors Weng X, Gong J, Chen Y, Wu T, Wang F, Yang S, Yuan Y, Luo G, Chen K, Hu L, Ma H, Wang P, Zhang QC*, Zhou X*, He C*

Abstract RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.
Journal Name
Nat Chem Biol 2020, In Press

Links
PDF

Authors Lu M, Zhang Z, Xue M, Zhao BS, Harder O, Li A, Liang X, Gao TZ, Xu Y, Zhou J, Feng Z, Niewiesk S, Peebles ME, He C*, Li J*

Abstract Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.
Journal Name
Nat Microbiol 2020, 5:584-598.

Links
PDF

Authors Liu J, Dou XY, Chen CY, Chen C, Liu C, Xu MM< Zhao SQ, Shen B, Gao YW*, Han DL*, He C*

Abstract N 6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. Here, we showed that knockout of the m6A writer Mettl3 or a nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-associated regulatory RNAs (carRNAs), including promoter-associated RNAs, enhancer RNAs and repeats RNAs. YTHDC1 facilitates decay of a subset of these m6A-modified RNAs, especially LINE1 elements, through the NEXT-mediated nuclear degradation. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results revealed that m6A on carRNAs can globally tune chromatin state and transcription.
Journal Name
Science 2020, 367:580-586.

Links
PDF

Authors Zhang Z, Zhan Q, Eckert M, Zhu A, Chryplewicz A, De Jesus DF, Ren D, Kulkarni RN, Lengyel E, He C*, Chen M*

Abstract Epitranscriptome profiling using MeRIP-seq is a powerful technique for in vivo functional studies of reversible RNA modifications. We develop RADAR, a comprehensive analytical tool for detecting differentially methylated loci in MeRIP-seq data. RADAR enables accurate identification of altered methylation sites by accommodating variability of pre-immunoprecipitation expression level and post-immunoprecipitation count using different strategies. In addition, it is compatible with complex study design when covariates need to be incorporated in the analysis. Through simulation and real dataset analyses, we show that RADAR leads to more accurate and reproducible differential methylation analysis results than alternatives, which is available at https://github.com/scottzijiezhang/RADAR
Journal Name
Genome Biol 2019, 20:294

Links
PDF

Authors Xue M, Zhao BS, Zhang Z, Lu M, Harder O, Chen P, Lu Z, Li A, Ma Y, Xu Y, Liang X, Zhou J, Niewiesk S, Peeples ME, He C, Li J

Abstract N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.
Journal Name
Nat Commun 2019, 10:4595

Links
PDF

Authors Zhou H, Rauch S, Dai Q, Cui X, Zhang Z, Nachtergaele S, Sepich C, He C, Dickinson BC

Abstract Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m1A sites and revealed hundreds of m1A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m1A biology.
Journal Name
Nat Methods 2019, 16:1281-1288

Links
PDF

Authors Zhou KI, Shi H, Lyu R, Wylder AC, Matuszek Ż, Pan JN, He C, Parisien M, Pan T

Abstract  N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.
Journal Name
Mol Cell 2019, 76:70-81

Links
PDF

Authors Zhang W, Eckwahl MJ, Zhou KI, Pan T

Abstract  =Pseudouridine (Ψ) is the most abundant RNA modification in cellular RNA present in tRNA/rRNA/snRNA and also in mRNA and long noncoding RNA (lncRNA). Elucidation of Ψ function in mRNA/lncRNA requires mapping and quantitative assessment of its modification fraction at single-base resolution. The most widely used Ψ mapping method for mRNA/lncRNA relies on its reaction with N-Cyclohexyl-N’-(2-morpholinoethyl)carbodiimide (CMC), forming an adduct with the Ψ base in RNA that is detectable by reverse transcription (RT) stops. However, this method has not produced consistent Ψ maps in mRNAs; furthermore, available protocols do not lend confidence to the estimation of Ψ fraction at specific sites, which is a crucial parameter for investigating the biological relevance of mRNA modifications. Here we develop a quantitative RT-PCR based method that can detect and quantify the modification fraction of target Ψ sites in mRNA/lncRNA, termed CMC-RT and ligation assisted PCR analysis of Ψ modification (CLAP). The method still relies on RT stop at a CMC-Ψ site, but uses site-specific ligation and PCR to generate two distinct PCR products in the same sample, corresponding to the modified and unmodified site, that are visualized by gel electrophoresis. CLAP not only requires a small amount of cellular RNA to validate Ψ sites but also determines the Ψ fraction semiquantitatively at target sites in mRNA/lncRNA. We determined the Ψ status of four mRNA sites and one lncRNA site whose modification fractions range from 30% to 84% in three human cell lines. Our method enables precise mapping and assessment of Ψ modification levels in low abundance cellular RNAs.
Journal Name
RNA 2019, 25: 1218-1225

Links
PDF

Authors Edens BM, Vissers C, Su J, Arumugam S, Xu Z, Shi H, Miller N, Rojas Ringeling F, Ming GL, He C, Song H*, Ma YC* 

Abstract  N6-methyladenosine (m6A) modification of mRNA is emerging as a vital mechanism regulating RNA function. Here, we show that fragile X mental retardation protein (FMRP) reads m6A to promote nuclear export of methylated mRNA targets during neural differentiation. Fmr1 knockout (KO) mice show delayed neural progenitor cell cycle progression and extended maintenance of proliferating neural progenitors into postnatal stages, phenocopying methyltransferase Mettl14 conditional KO (cKO) mice that have no m6A modification. RNA-seq and m6A-seq reveal that both Mettl14cKO and Fmr1KO lead to the nuclear retention of m6A-modified FMRP target mRNAs regulating neural differentiation, indicating that both m6A and FMRP are required for the nuclear export of methylated target mRNAs. FMRP preferentially binds m6A-modified RNAs to facilitate their nuclear export through CRM1. The nuclear retention defect can be mitigated by wild-type but not nuclear export-deficient FMRP, establishing a critical role for FMRP in mediating m6A-dependent mRNA nuclear export during neural differentiation.
Journal Name
Cell Rep 2019, 28: 845-854

Links
PDF

Authors De Jesus DF, Zhang Z, Kahraman S, Brown NK, Chen M, Hu J, Gupta MK, He C*, Kulkarni RN*

Abstract  The regulation of islet cell biology is critical for glucose homeostasis1.N6 -methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification in mammals2. Here we report that the m6A landscape segregates human type 2 diabetes (T2D) islets from controls significantly better than the transcriptome and that m6A is vital for β-cell biology. m6A-sequencing in human T2D islets reveals several hypomethylated transcripts involved in cell-cycle progression, insulin secretion, and the Insulin/IGF1-AKT-PDX1 pathway. Depletion of m6A levels in EndoC-βH1 induces cell-cycle arrest and impairs insulin secretion by decreasing AKT phosphorylation and PDX1 protein levels. β-cell specific Mettl14 knock-out mice, which display reduced m6A levels, mimic the islet phenotype in human T2D with early diabetes onset and mortality due to decreased β-cell proliferation and insulin degranulation. Our data underscore the significance of RNA methylation in regulating human β-cell biology, and provide a rationale for potential therapeutic targeting of m6A modulators to preserve β-cell survival and function in diabetes.
Journal Name
Nat Metab 2019, 8: 765-774

Links
PDF

Authors Zhang LS, Liu C, Ma H, Dai Q, Sun HL, Luo G, Zhang Z, Zhang L, Hu L, Dong X, He C

Abstract  N7-methylguanosine (m7G) is a positively charged, essential modification at the 5′ cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.
Journal Name
Mol Cell 2019, 74: 1304-1316

Links
PDF

Authors Shi H, Wei J, He C

Abstract  Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification “effectors,” including enzymes (“writers” and “erasers”) that alter the modification level and binding proteins (“readers”) that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors. We emphasize the importance of context for RNA modification regulation and function.
Journal Name
Mol Cell 2019, 74: 640-650

Links
PDF

Authors Rauch S, He E, Srienc M, Zhou H, Zhang Z, Dickinson BC

Abstract  Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.
Journal Name
Cell 2019, 178: 122-134

Links
PDF

Authors Huang H, Weng H, Zhou K, Wu T, Zhao BS, Sun M, Chen Z, Deng X, Xiao G, Auer F, Klemm L, Wu H, Zuo Z, Qin X, Dong Y, Zhou Y, Qin H, Tao S, Du J, Liu J, Lu Z, Yin H, Mesquita A, Yuan C. L, Hu YC, Sun W, Su R, Dong L, Shen C, Li C, Qing Y, Jiang X, Wu X, Sun M, Guan JL, Qu L, Wei M, Müschen M, Huang G, He C, Yang J, Chen J

Abstract  DNA and histone modifications have notable effects on gene expression. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation and has crucial roles in various normal and pathological processes. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.
Journal Name
Nature 2019, 567: 414-419

Links
PDF

Authors  Liu J, Harada BT, He C

Abstract  As the most abundant mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) has recently emerged as an important regulator of gene expression. m6A modification can be deposited by m6A methyltransferases, removed by m6A demethylases, and recognized by different reader proteins. Numerous lines of evidence have shown that m6A methylation plays critical roles regulating gene expression in development and disease. In this review, we summarize the molecular and cellular function of m6A and highlight some key results which demonstrate the role of m6A in various cancers. Finally, we discuss future directions for research into m6A and its effects in cancer and the potential for targeting RNA modification in cancer treatment.
Journal Name
Trends Cell Biol 2019, In Press

Links
PDF

Journal Name
Biochemistry 2018, 57: 1055-56

Links
PDF

Journal Name
Neuron 2018, 97:313-325

Links
PDF

Journal Name
Cell 2018, 172:90-105

Links
PDF

Journal Name
Nat. Methods. 2017, 14:695-698

Links
PDF

Journal Name
Human Mol. Genet. 2017, 26:2398-2411

Links
PDF

Journal Name
Cell. 2017, 169:1187-1200

Links
PDF

Authors
Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C 

Abstract
We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO

Journal Name
Nat Chem Biol. 2011, 7(12):885-7

Links
https://www.nature.com/nchembio/journal/v7/n12/full/nchembio.687.html

Authors
Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C 

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.

Journal Name
Nature. 2014, 505(7481):117-20

Links
https://www.nature.com/nature/journal/v505/n7481/full/nature12730.html

Authors
Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, Dai Q, Chen W, He C.:  

Abstract
N(6)-methyladenosine (m(6)A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m(6)A RNA methylation. Together with METTL3, the only previously known m(6)A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m(6)A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Journal Name
Nat Chem Biol. 2014, 10(2):93-5

Links
https://www.nature.com/nchembio/journal/v10/n2/full/nchembio.1432.html

Authors
Xu C, Wang X, Liu K, Roundtree IA, Tempel W, Li Y, Lu Z, He C, Min J 

Abstract
N(6)-methyladenosine (m(6)A) is the most abundant internal modification of nearly all eukaryotic mRNAs and has recently been reported to be recognized by the YTH domain family proteins. Here we present the crystal structures of the YTH domain of YTHDC1, a member of the YTH domain family, and its complex with an m(6)A-containing RNA. Our structural studies, together with transcriptome-wide identification of YTHDC1-binding sites and biochemical experiments, not only reveal the specific mode of m(6)A-YTH binding but also explain the preferential recognition of the GG(m(6)A)C sequences by YTHDC1.

Journal Name
Nat Chem Biol. 2014, 10(11):927-9

Links
https://www.nature.com/nchembio/journal/v10/n11/full/nchembio.1654.html

Authors
Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C 

Abstract
N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms

Journal Name
Cell. 2015, 161(6):1388-99

Links
https://www.sciencedirect.com/science/article/pii/S0092867415005620

Authors
 Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T.

Abstract
RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism ‘the m(6)A-switch’. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology

Journal Name
Nature. 2015,  518(7540):560-4

Links
https://www.nature.com/nature/journal/v518/n7540/full/nature14234.html

Authors
Luo GZ, MacQueen A, Zheng G, Duan H, Dore LC, Lu Z, Liu J, Chen K, Jia G, Bergelson J, He C.  

Abstract
Recent discoveries of reversible N(6)-methyladenosine (m(6)A) methylation on messenger RNA (mRNA) and mapping of m(6)A methylomes in mammals and yeast have revealed potential regulatory functions of this RNA modification. In plants, defects in m(6)A methyltransferase cause an embryo-lethal phenotype, suggesting a critical role of m(6)A in plant development. Here, we profile m(6)A transcriptome-wide in two accessions of Arabidopsis thaliana and reveal that m(6)A is a highly conserved modification of mRNA in plants. Distinct from mammals, m(6)A in A. thaliana is enriched not only around the stop codon and within 3′-untranslated regions, but also around the start codon. Gene ontology analysis indicates that the unique distribution pattern of m(6)A in A. thaliana is associated with plant-specific pathways involving the chloroplast. We also discover a positive correlation between m(6)A deposition and mRNA abundance, suggesting a regulatory role of m(6)A in plant gene expression.

Journal Name
Nat Commun. 2014, 5:5630

Links
https://www.nature.com/articles/ncomms6630

Authors
Fu Y, Jia G, Pang X, Wang RN, Wang X, Li CJ, Smemo S, Dai Q, Bailey KA, Nobrega MA, Han KL, Cui Q, He C 

Abstract
N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

Journal Name
Nat Commun. 2013, 4:1798

Links
https://www.nature.com/articles/ncomms2822

Authors
Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC, Zheng G, Pan T, Solomon O, Eyal E, Hershkovitz V, Han D, Doré LC, Amariglio N, Rechavi G, He C 

Abstract
Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Journal Name
Nature. 2016,  530(7591):441-6

Links
https://www.nature.com/nature/journal/v530/n7591/full/nature16998.html

Authors
Liu F, Clark W, Luo GZ, Wang XY, Fu Y, Wei J-B, Wang X, Hao ZY, Dai Q, Zheng QQ, Ma HH, Han D, Evans M, Klungland A, Pan T, He, C.:  

Abstract
tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

Journal Name
Cell. 2016

Links
https://www.sciencedirect.com/science/article/pii/S009286741631323X

Authors
Liu N, Parisien M, Dai Q, Zheng G, He C, Pan T.

Abstract
N(6)-methyladenosine (m(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m(6)A demethylases and cell-type and cell-state-dependent m(6)A patterns indicate that m(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(6)A modification. We applied the method to determine the m(6)A status at several sites in two human lncRNAs and three human mRNAs and found that m(6)A fraction varies between 6% and 80% among these sites. We also found that many m(6)A candidate sites in these RNAs are however not modified. The precise determination of m(6)A status in a long noncoding RNA also enables the identification of an m(6)A-containing RNA structural motif.

Journal Name
RNA. 2013, 19(12):1848-56

Links
https://rnajournal.cshlp.org/content/19/12/1848.long

Authors
Chen K, Lu Z, Wang X, Fu Y, Luo GZ, Liu N, Han D, Dominissini D, Dai Q, Pan T, He C 

Abstract
N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

Journal Name

Angew Chem Int Ed Engl. 2015, 54(5):1587-90.

Links
https://onlinelibrary.wiley.com/doi/10.1002/anie.201410647/abstract;jsessionid=B54843FA58DCE36756771022F0AC26F8.f03t04

Authors
Zheng G, Qin Y, Clark WC, Dai Q, Yi C, He C, Lambowitz AM, Pan T 

Abstract
Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.

Journal Name
Nat Methods. 2015, 12(9):835-7.

Links
https://www.nature.com/nmeth/journal/v12/n9/full/nmeth.3478.html

Authors Clark WC, Evans ME, Dominissini D, Zheng G, Pan T  

Abstract Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative “Modification Index” (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

Journal Name RNA, 2016

Links https://rnajournal.cshlp.org/content/22/11/1771.long