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RNA pulldown-MS, Dlg4 RNA in mouse brain

ABSTRACT: Goal was to identify RNA-binding proteins with a preference for m6A-modified RNA using an RNA bait generated from a known m6A site in the Dlg4 (PSD-95) mRNA 3`-UTR. By comparison of differential enrichment between m6A-RNA pulldown fractions and A-RNA or no RNA fractions, we can identify m6A-binding proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Kate Meyer

LAB HEAD: Kate Meyer

PROVIDER: PXD034159 | Pride | 2022-10-13

REPOSITORIES: Pride

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Publications

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

Choi Seung H SH Flamand Mathieu N MN Liu Bei B Zhu Huanyu H Hu Meghan M Wang Melanie M Sewell Jonathon J Holley Christopher L CL Al-Hashimi Hashim M HM Meyer Kate D KD

Cell reports 20220801 9

N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that ...[more]

PMID: 36044854

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RNA pulldown-MS, Dlg4 RNA in mHippoE-2 cells

Project description:Goal was to identify RNA-binding proteins with a preference for m6A-modified RNA using an RNA bait generated from a known m6A site in the Dlg4 (PSD-95) mRNA 3`-UTR. By comparison of differential enrichment between m6A-RNA pulldown fractions and A-RNA or no RNA fractions, we can identify m6A-binding proteins.

2022-10-13 | PXD034161 | Pride

Identification of proteins specifically binding to ac4C RNA probes

Project description:As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.

2021-07-20 | PXD027014 | Pride

The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia

Project description:In this work we dissect the functional role of the HOXB-AS3 long non coding RNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 was found to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

2019-10-16 | PXD015520 | Pride

Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3' UTRs and Near Stop Codons (OLD)

Project description:Here, we use a novel technique for locating regions of N6-adenosine methylation (m6A) throughout the transcriptome and present a profile of m6A sites in the mouse brain. Our use of methylated RNA immunoprecipitation combined with RNA-seq (MeRIP-Seq) identifies thousands of RNAs which contain m6A sites. In addition, we find that regions of m6A formation are particularly enriched near stop codons, which might provide clues into the potential funciton of this highly prevalent RNA modificaiton. Examination of m6A sites in murine brain RNA and human embryonic kidney cells.

2012-12-31 | E-GEOD-34734 | biostudies-arrayexpress

Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

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 Î±-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

Adenosine methylation is widespread throughout the transcriptome and prominent within 3' UTRs

2012-05-16 | E-GEOD-29714 | biostudies-arrayexpress

m6A RNA modification controls cell fate transition in mammalian embryonic stem cells (RNA methylation)

Project description:Here we determine the map of RNA methylation (m6A) in mouse embrionic stem cells, and Mettl3 knock out cells Examination of m6A modification sites on the transcriptome of mouse Embryonic stem cells and Embryonic Mettl3 knock out cells, using a m6A specific antibody.

2014-10-16 | E-GEOD-52662 | biostudies-arrayexpress

m6A RNA Methylation Is Regulated by MicroRNAs and Promotes Reprogramming to Pluripotency

Project description:N6-methyladenosine (m6A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m6A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m6A, especially gene- and cell-type-specific m6A mRNA modifications. We also show that microRNAs (miRNAs) regulate m6A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m6A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m6A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m6A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m6A formation of mRNAs and provide a foundation for future functional studies of m6A modification in cell reprogramming. m6A-seq in ESC, iPSC, NSC and sertoli cells.

2015-03-10 | E-GEOD-52125 | biostudies-arrayexpress

m6A RNA modification controls cell fate transition in mammalian embryonic stem cells

Project description:N6-methyl-adenosine (m6A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m6A by mapping the m6A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m6A modification, including transcripts encoding core pluripotency transcription factors. m6A is enriched over 3M-bM-^@M-^Y untranslated regions at defined sequence motifs, and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m6A methylases, led to m6A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESCM-bM-^@M-^Ys exit from self-renewal towards differentiation into several lineages in vitro and in vivo. Thus, m6A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages. Examing m6A modification differences in two different cell types

2014-10-20 | E-GEOD-52600 | biostudies-arrayexpress

Structure and Thermodynamics of N6-methyladenosine in RNA: a Spring-Loaded Base Modification

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