Aaron Gitler, Postdoctoral Faculty Sponsor
METTL14 is a chromatin regulator independent of its RNA N-6-methyladenosine methyltransferase activity
PROTEIN & CELL
METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.
View details for DOI 10.1093/procel/pwad009
View details for Web of Science ID 000964216400001
View details for PubMedID 37030005
Exon architecture controls mRNA m6A suppression and gene expression.
Science (New York, N.Y.)
2023; 379 (6633): 677-682
N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.
View details for DOI 10.1126/science.abj9090
View details for PubMedID 36705538
View details for PubMedCentralID PMC9990141
m7G-quant-seq: Quantitative Detection of RNA Internal N7-Methylguanosine
ACS CHEMICAL BIOLOGY
2022; 17 (12): 3306-3312
Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m7G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m7G-quant-seq, a quantitative method that accurately detects internal m7G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m7G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m7G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m7G methylation level. We detected internal m7G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m7G methylation stoichiometry. m7G-quant-seq could be applied to monitor the tRNA m7G methylation level change in diverse biological processes.
View details for DOI 10.1021/acschembio.2c00792
View details for Web of Science ID 000889577500001
View details for PubMedID 36398936
View details for PubMedCentralID PMC9764283
ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing
NATURE CELL BIOLOGY
2021; 23 (7): 684-+
Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.
View details for DOI 10.1038/s41556-021-00709-7
View details for Web of Science ID 000672276000007
View details for PubMedID 34253897
View details for PubMedCentralID PMC8716185
QSER1 protects DNA methylation valleys from de novo methylation
2021; 372 (6538): 146-+
DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.
View details for DOI 10.1126/science.abd0875
View details for Web of Science ID 000639216800034
View details for PubMedID 33833093
View details for PubMedCentralID PMC8185639
DNA 5-Methylcytosine-Specific Amplification and Sequencing.
Journal of the American Chemical Society
DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.
View details for DOI 10.1021/jacs.9b12707
View details for PubMedID 32077696
N-6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription
2020; 367 (6477): 580-+
N 6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. In this work, we show that knockout of the m6A writer Mettl3 or the 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 repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, especially elements of the long interspersed element-1 family, through the nuclear exosome targeting-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 reveal that m6A on carRNAs can globally tune chromatin state and transcription.
View details for DOI 10.1126/science.aay6018
View details for Web of Science ID 000512377600043
View details for PubMedID 31949099
View details for PubMedCentralID PMC7213019
Transcriptome-wide Mapping of Internal N-7-Methylguanosine Methylome in Mammalian mRNA
2019; 74 (6): 1304-+
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.
View details for DOI 10.1016/j.molcel.2019.03.036
View details for Web of Science ID 000472231600019
View details for PubMedID 31031084
View details for PubMedCentralID PMC6588483
N-6-Methyladenosine- binding proteins suppress HIV-1 infectivity and viral production
JOURNAL OF BIOLOGICAL CHEMISTRY
2018; 293 (34): 12992-13005
The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to m6A-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m6A modification of HIV-1 RNA affects viral replication.
View details for DOI 10.1074/jbc.RA118.004215
View details for Web of Science ID 000442730200002
View details for PubMedID 29976753
View details for PubMedCentralID PMC6109920
Recognition of RNA N-6- methyladenosine by IGF2BP proteins enhances mRNA stability and translation
NATURE CELL BIOLOGY
2018; 20 (3): 285-+
N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.
View details for DOI 10.1038/s41556-018-0045-z
View details for Web of Science ID 000426059400011
View details for PubMedID 29476152
View details for PubMedCentralID PMC5826585
YTHDF3 facilitates translation and decay of N-6-methyladenosine-modified RNA
2017; 27 (3): 315-328
N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and tissue development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and decay, via the recognition by selective binding proteins. In the cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH (YT521-B homology) domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated through YTHDF2. Cells deficient in all three YTHDF proteins experience the most dramatic accumulation of m6A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m6A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m6A RNA methylation.
View details for DOI 10.1038/cr.2017.15
View details for Web of Science ID 000396265500005
View details for PubMedCentralID PMC5339834
Enantioselective Total Syntheses of Various Amphilectane and Serrulatane Diterpenoids via Cope Rearrangements
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2016; 138 (19): 6261-6270
Ampilectane and serrulatane natural products are structurally and stereochemically complex compounds that display various potent pharmacological activities ranging from anti-inflammatory to antituberculosis. A general synthetic route toward this family of natural products has been developed, which accomplished a number of amphilectane and serrulatane natural products. The key step employed a stereoselective Cope rearrangement either promoted by gold catalysis or thermal conditions, while a regioselective gold-catalyzed 6-endo-dig cyclization was optimized to afford a precursor. The preparation of the chiral β-ketoester as a starting material was established via an optimized asymmetric 1,4-addition followed by trapping with Mander's reagent, and this initially installed stereogenic center provided good control in the subsequent introduction of all the other stereocenters. A rarely investigated one-pot conversion of α-pyrone into phenol was also examined to enable the syntheses. DFT calculations explain the high stereoselectivity of the Cope rearrangement of the intermediate that eventually led to amphilectolide and caribenol A.
View details for DOI 10.1021/jacs.6b02624
View details for Web of Science ID 000376331000028
View details for PubMedID 27115064
Photo-induced coupling reactions of tetrazoles with carboxylic acids in aqueous solution: application in protein labelling
2016; 52 (25): 4702-4705
The photo-induced reactions of diaryltetrazoles with carboxylic acids in aqueous solution were investigated. Besides measuring the apparent second-order rate constant and evaluating the functional group compatibility of these reactions, we further incorporated the tetrazoles into SAHA, leading to a new active-site-directed probe for labelling HDACs in both cell lysates and living cells.
View details for DOI 10.1039/c5cc10445a
View details for Web of Science ID 000372257400025
View details for PubMedID 26953773