Structure and Thermodynamics of N6-methyladenosine in RNA: a Spring-Loaded Base Modification
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE65380 | GEO | 2015/01/29
SECONDARY ACCESSION(S): PRJNA273856
REPOSITORIES: GEO
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Project description:We determine m6A modification sites in GM12878 cells. Examination of m6A modification sites on the transcriptome of GM12878 cells, using a m6A-specific antibody.
2015-01-29 | E-GEOD-65380 | biostudies-arrayexpress
Project description:N(6)-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)A-U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the Watson-Crick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m(6)A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m(6)A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.
| S-EPMC4405242 | biostudies-literature
Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down
2015-06-09 | E-GEOD-63591 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and α-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates
2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress
Project description:Identification of m6A modification sites on circRNAs in Hela cells.
2019-08-29 | GSE116007 | GEO
Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown
2015-02-27 | E-GEOD-56010 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is one of the most popular RNA modifications, which is widely found in messenger RNAs (mRNAs) and non-coding RNA like long no-coding RNA (lncRNAs) and circular RNA (circRNAs).In our study,we provide m6A landscape of human ameloblastoma, which expands the understanding of m6A modifications and uncovers regulation of lncRNAs and circRNAs through m6A modification in ameloblastoma.
2020-10-08 | GSE156886 | GEO
Project description:N6-methyladenosine (m6A) is the most common prevalent internal modifications found in many of the eukaryotic mRNA and plays an important role in RNA metabolism including pre-mRNA processing, mRNA stability, RNA splicing, RNA export and nuclear retention. Serrate is a component of the Dicer complex, plays as a key factor in RNA metabolism. We here reported SERRATE acts as a key regulator of mRNA m6A modification in Arabidopsis. Loss function of SE results in significant global m6A level reduction in the se-1 mutant comparing to the Col-0. SE positively regulates the transcription of MTA and MTB. In addition, SE physically interacts and functionally works with the m6A writer complex MTA and MTB which affects the binding of m6A methylase to mRNA. Taking together, our data provides a molecular framework that SE modulates m6A mRNA modification in Arabidopsis.
2023-11-01 | GSE241248 | GEO
Project description:N6-methyladenosine (m6A) modification of messenger RNAs (mRNAs) is a pivotal mechanism controlling mRNA fate in cells. RNA m6A modification is regulated by the functional balance between methyltransferases and demethylases. Here we demonstrated that FTO-IT1 enhancer RNA (eRNA), a long non-coding RNA (lncRNA) transcribed from the last intron of FTO gene is significantly upregulated in CRPC and aggressive tumors compared to primary tumors. FTO-IT1 knockout by CRISPR/Cas9 almost completely blocks growth and G1-S cell cycle transition of both androgen-sensitive and castration-resistant prostate cancer cells. Meanwhile, the mRNA m6A was dramatically increased in FTO-IT knockout PCa cells and we identified FTO-IT1 as a binding partner of FTO. From m6A-seq, we unexpectedly found hypermethylated m6A associated with upregulated levels of the mRNAs for p53 signaling pathway genes in 22Rv1 prostate cancer cells. Mechanistic study showed that FTO-IT1 recruits FTO to the P53 target mRNA to promote their m6A demethylation, which leads to their degradation.
2023-07-09 | GSE189465 | GEO
Project description:Although internal PolyA RNA modification N6-methyladenosine (m6A) plays essential roles in diverse biological processes, technology to detect precise m6A sites at transcriptome-wide scale is lacking. Here, we discovered that m6A interferes A (Adenine) – U (Uracil) or A-T (Thymidine) pairing. Based on differential hybridization between methylated vs. unmethylated RNAs to a DNA probe, we developed tiling microarray to pinpoint m6A sites in mouse transcriptome. We validated some of the identified sites and provided evidence to suggest that one functional mechanism of m6A is to block small RNA targeting to methylated mRNA. We designed a custom tiling array with to examine the precise location of m6A within meRIP-seq peaks from mouse embryonic stem cells determined in our previous publication (Wang et al., 2014). Each custom two-channel Agilent tiling array harbors 947,952 probes. Each probe is 25 nucleotides (nt), and any two adjacent probes in the genomic coordinate overlap each other by 19 nt. The Cy5 or red channel corresponds to Mettl14 knockout (M14) or DZA mutant mESC cell line, and Cy3 or green channel is associated with wild type cell line treated with scramble hairpin (SCR). Thus, in principle a higher Cy5/Cy3 signal for each probe reflects an increased hybridization to the oligonucleotide due to de-methylation of a particular RNA molecule in M14 or DZA condition relative to the SCR control. Moreover, we employed additional arrays with both channels dedicated for M14 as an external control for technical difference between the Cy5 and Cy3 dye (details below). For each comparison, we have three biological replicates, and therefore there are 9 tiling arrays in total (i.e., 3 arrays for M14 vs SCR, 3 arrays for DZA vs SCR, and 3 arrays for M14 vs M14).
2015-09-08 | E-GEOD-62530 | biostudies-arrayexpress