Improved methods for deamination-based m6A detection
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE196416 | GEO | 2022/05/11
REPOSITORIES: GEO
Similar Datasets
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
2014-09-18 | E-GEOD-58352 | biostudies-arrayexpress
Project description:Improved methods for deamination-based m6A detection
| PRJNA804865 | ENA
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader.
2014-09-18 | GSE58352 | GEO
Project description:m6A is a widespread RNA modification which plays important roles in the regulation of gene expression. Methods for the global detection of m6A rely on immunoprecipitation of methylated transcripts using m6A antibodies. However, these methods are costly and require large amounts of input RNA, making them prohibitive for many experiments. Here, we describe DART-seq, an antibody-free method for m6A detection which enables transcriptome-wide mapping of m6A residues using low amounts of input material. DART-seq can be used to obtain global m6A maps using as little as 10 nanograms of total RNA and at single-molecule resolution. This method offers several improvements over current techniques and will facilitate detection of m6A in limiting cell and tissue types.
2019-09-23 | GSE125780 | GEO
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNA of all higher eukaryotes. Here we present that m6A is selectively recognized by human YTH domain family (YTHDF2) protein to regulate mRNA degradation. By using crosslinking and immunoprecipitation, we have identified over 4000 substrate RNA of YTHDF2 with conserved core motif of G(m6A)C. We further estabilshed the role of YTHDF2 in RNA metabolism by a combination of ribosome profiling, RNA sequencing, m6A level quantification and cell-based imaging: the C-terminal domain of YTHDF2 selectively binds to m6A of mRNA and the N-terminal domain is responsive for localizing mRNA from translatable pool to processing body where mRNA decay occurs. PAR-CLIP and RIP was used to identify YTHDF2 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF2 siRNA knock-down
2013-11-29 | E-GEOD-49339 | biostudies-arrayexpress
Project description:N 6-methyladenosine (m6A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m6A has been enabled by the identification of m6A sites transcriptome-wide. However, global m6A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m6A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m6A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m6A sites, therefore enabling single nucleotide-resolution m6A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m6A detection and will facilitate the mapping of m6A in samples not previously amenable to global m6A profiling.
| S-EPMC9092492 | biostudies-literature
Project description:We used DART-seq to map m6A methylation of RNA in single HEK293T cells. We also used DART-seq to map m6A from bulk RNA from HEK293T cells. Using the 10X Genomics and SMART-seq2 platforms, we sequenced a total of 19,533 experimental and control cells using the 10X Genomics platform, and 1,471 experimental and control cells using SMART-seq2. We then used a Bullseye, a computational pipeline developed within the lab, to identify m6A sites from the C-to-U mutations in bulk and single-cell datasets. We find that most m6A methylation is highly heterogenous from cell-to-cell. RNAs containing m6A methylation, are infrequently methylated, and that most individual sites are rarely methylated within the population. Additionally, we are able to identify differentially methylated RNAs in different cellular states from within a single population, and use m6A methylation information to perform clustering of single cells to find a source of novel cellular heterogeneity.
2022-01-25 | GSE180954 | GEO
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:Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the recent finding of FTO?s (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggested a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA ?epimark? remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells. The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae, we also sought to test whether it can be induced under non-standard sporulation conditions. We find associations between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment. GeneChip analyses were performed to investigate the distribution of early meiotic transcripts (3hours) on different polysome and monosome fractions
2015-08-12 | E-GEOD-68435 | biostudies-arrayexpress
Project description:Heat shock induces a cell response leading to profound changes in genome expression. Recently, N6-methyladenosine (m6A) RNA modification has been implicated in this response, but with limited information of its role in the heat-induced reprograming of gene expression. Most of m6A molecular and cellular functions rely on m6A readers and the best characterized m6A readers are members of the YTH-domain-containing protein family present from yeast to humans. To investigate the function of the nuclear m6A reader YTHDC1, we characterized its binding partners.
2023-01-11 | PXD037414 | Pride