Publications

CEGS-supported Publications

Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis

Authors Li X, Yao B, Chen L, Kang Y, Li Y, Cheng Y, Li L, Wang Z, Wang M, Pan F, Dai Q, Zhang W, Wu H, Shu Q, Qin Z, He C, Xu M, Jin P

Abstract
Emerging evidence suggests that active DNA demethylation machinery plays important epigenetic roles in mammalian adult neurogenesis; however, the precise molecular mechanisms and critical functional players of DNA demethylation in this process remain largely unexplored. Ten-eleven translocation (Tet) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and its downstream derivatives. Here we show that 5hmC is elevated during the differentiation of adult neural stem cells (aNSCs), and Tet2 is primarily responsible for modulating 5hmC dynamics. Depletion of Tet2 leads to increased aNSC proliferation and reduced differentiation in vitro and in vivo. Genome-wide transcriptional analyses reveal important epigenetic roles of Tet2 in maintaining the transcriptome landscape related to neurogenesis. Mechanistically, transcription factor forkhead box O3 (Foxo3a) physically interacts with Tet2 and regulates the expression of genes related to aNSC proliferation. These data together establish an important role for the Tet2-Foxo3a axis in epigenetically regulating critical genes in aNSCs during adult neurogenesis.

Journal Name
Nat Commun. 2017, 8:15903

Links
PDF

Selective enzymatic demethylation of N2, N2-dimethylguanosine in RNA and its application in high-throughput tRNA sequencing

Authors Dai Q, Zheng G, Schwartz MH, Clark WC, Pan T

Abstract
The abundant Watson-Crick face methylations in biological RNAs such as N1-methyladenosine (m1A), N1-methylguanosine (m1G), N3-methylcytosine (m3C), and N2,N2-dimethylguanosine (m2 2G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m1A, m1G, m3C modifications in transfer RNA (tRNA), but they work poorly on m2 2G. Here we report the design and evaluation of a series of AlkB mutants against m2 2G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m2 2G modification to N2-methylguanosine (m2G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Journal Name
Angew Chem Int Ed Engl. 2017, 56(18):5017-5020

Links
PDF

m6A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells

Authors Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G, Sun G, Lu Z, Huang Y, Yang CG, Riggs AD, He C, Shi Y 

Abstract
m6A RNA modifications play critical roles in important biological processes. However, the functions of N6-methyladenosine (m6A) mRNA modification in cancer biology and cancer stem cells remain largely unknown. Here, we show that m6A mRNA modification is critical for glioblastoma stem cell (GSC) self-renewal and tumorigenesis. Knockdown of METTL3 or METTL14, key components of the methyltransferase complex, dramatically promotes human GSC growth, self-renewal, and tumorigenesis. In contrast, overexpression of METTL3 or inhibition of the demethylase FTO suppresses GSC growth and self-renewal. Moreover, inhibition of FTO suppresses tumor progression and prolongs lifespan of GSC-grafted mice substantially. m6A sequencing reveals that knockdown of METTL3 or METTL14 induced changes in mRNA m6A enrichment and altered mRNA expression of genes (e.g., ADAM19) with critical biological functions in GSCs. In summary, this study identifies the m6A mRNA methylation machinery as promising therapeutic targets for glioblastoma.

Journal Name
Cell Rep. 2017, 18(11):2622-2634

Links
PDF

Advances in Zika virus researh: stem cell models, challenges, and opportunities

Authors Ming GL, Tang H, Song H

Abstract
The re-emergence of Zika virus (ZIKV) and its suspected link with various disorders in newborns and adults led the World Health Organization to declare a global health emergency. In response, the stem cell field quickly established platforms for modeling ZIKV exposure using human pluripotent stem cell-derived neural progenitors and brain organoids, fetal tissues, and animal models. These efforts provided significant insight into cellular targets, pathogenesis, and underlying biological mechanisms of ZIKV infection as well as platforms for drug testing. Here we review the remarkable progress in stem cell-based ZIKV research and discuss current challenges and future opportunities.

Journal Name
Cell Stem Cell. 2016, 19(6):690-702

Links
PDF

Past Publications

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility

Authors Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vågbø CB, Shi Y, Wang WL, Song SH, Lu Z, Bosmans RP, Dai Q, Hao YJ, Yang X, Zhao WM, Tong WM, Wang XJ, Bogdan F, Furu K, Fu Y, Jia G, Zhao X, Liu J, Krokan HE, Klungland A, Yang YG, He C

Abstract
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Journal Name
Mol Cell. 2013, 49(1):18-29

Links
http://www.sciencedirect.com/science/article/pii/S1097276512008921

N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

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

N6-methyladenosine-dependent regulation of messenger RNA stability

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

A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

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

Structural basis for selective binding of m 6 A RNA by the YTHDC1 YTH domain

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

N6-methyladenosine modulates messenger RNA translation efficiency

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
http://www.sciencedirect.com/science/article/pii/S0092867415005620

N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

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

Unique features of the m6A methylome in Arabidopsis thaliana

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
http://www.nature.com/articles/ncomms6630

FTO-mediated formation of N 6 -hydroxymethyladenosine and N 6 -formyladenosine in mammalian RNA

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
http://www.nature.com/articles/ncomms2822

N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

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

ALKBH1-mediated tRNA demethylation regulates translation

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
http://www.sciencedirect.com/science/article/pii/S009286741631323X

Probing N6 -methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

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
http://rnajournal.cshlp.org/content/19/12/1848.long

High-resolution N6 -methyladenosine (m6A) map using photo-crosslinking-assisted m6A sequencing

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

Efficient and quantitative high-throughput tRNA sequencing

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

tRNA base methylation identification and quantification via high-throughput sequencing

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 http://rnajournal.cshlp.org/content/22/11/1771.long