Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Dominissini, Dan; Moshitch-Moshkovitz, Sharon; Salmon‐Divon, Mali; Amariglio, Ninette; Rechavi, Gideon

doi:10.1038/nprot.2012.148

Cited by 529 publications

(527 citation statements)

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“…Libraries were prepared from immunoprecipitated m 6 A‐containing RNAs and then subjected to next‐generation sequencing. As it relies on RNA fragmentation, its resolution is around 100‐200 nt, making it hard to determine the precise locations of m 6 A in RNA and losing much stoichiometry information 6, 36. In order to achieve a higher resolution, many methods are developed, such as PA‐m 6 A‐Seq (photo‐crosslinking‐assisted m 6 A‐sequencing) and SCARLET (site‐specific cleavage and radioactive‐labelling followed by ligation‐assisted extraction and thin‐layer chromatography) 37, 38.…”

Section: The Discovery Of M6a and Its Functionmentioning

confidence: 99%

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

Wang

et al. 2018

J Cellular Molecular Medi

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As the most abundant and reversible RNA modification in eukaryotic cells, m6A triggers a new layer of epi‐transcription. M6A modification occurs through a methylation process modified by “writers” complexes, reversed by “erasers”, and exerts its role depending on various “readers”. Emerging evidence shows that there is a strong association between m6A and human diseases, especially cancers. Herein, we review bi‐aspects of m6A in regulating cancers mediated by the m6A‐associated proteins, which exert vital and specific roles in the development of various cancers. Generally, the m6A modification performs promotion or inhibition functions (dual role) in tumorigenesis and progression of various cancers, which suggests a new concept in cancer regulations. In addition, m6A‐targeted therapies including competitive antagonists of m6A‐associated proteins may provide a new tumour intervention in the future.

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Section: The Discovery Of M6a and Its Functionmentioning

confidence: 99%

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

Wang

et al. 2018

J Cellular Molecular Medi

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“…P-9005; Epigentek) according to the manufacturer's instructions. To measure m 6 A + NANOG mRNA levels, m 6 A immunoprecipitation was performed as described before (47). A 1-μg aliquot of m 6 A antibody was conjugated to protein A-agarose slurry (Millipore) overnight at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m ⁶ A-demethylation of NANOG mRNA

Zhang

Samanta

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

834

818

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N 6 -methyladenosine (m 6 A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m 6 A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxiainducible factor (HIF)-1α-and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m 6 A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m 6 A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF-and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O 2 , HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.hypoxia-inducible factors | metastasis | pluripotency factors | self-renewal | tumorigenesis B reast cancer has the highest incidence of all cancers affecting women (1). It is also the leading cause of cancer-related death for women worldwide (2). Although progress has been made in treating early-stage breast cancer, there is no effective strategy to prevent or treat metastasis, which is the major cause of breast cancer-related mortality (3). Within breast cancers, a small subpopulation of cells, which are designated as tumor-initiating cells or breast cancer stem cells (BCSCs), have the unique property of generating daughter BCSCs, which are capable of infinite proliferation through self-renewal, and transient amplifying cells, which are capable of a limited number of cell divisions and give rise to differentiated breast cancer cells (4, 5). Only BCSCs are capable of forming a secondary (recurrent or metastatic) tumor (5, 6). As in the case of ES cells (ESCs) (7), the BCSC phenotype is specified by the expression of core pluripotency factors, including Kruppel-like factor 4 (KLF4), Octamer-binding transcription factor 4 (OCT4), SRY-box 2 (SOX2), and NANOG (8-12). Delineation of the molecular mechanisms that regulate the BCSC phenotype is needed to better understand metastasis and design more effective therapies.Intratumoral hypoxia is a common finding in advanced cancers. The median partial pressure of oxygen (pO 2 ) within primary breast cancers is 10 mm Hg [1.4% (vol/vol) O 2 ], compared with 65 mm Hg [9.3% (vol/vol) O 2 ] in normal breast tissue (13). Hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 are heterodimeric transcriptional activators, consisting of an O 2 -regu...

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“…SRR3667693). A-seq data were analyzed as described in the protocol by Dominissini et al (18) with some modifications. Briefly, the RNA-seq reads were mapped to the mRNAs of X. laevis collected by Xenbase (19) using Bowtie software (40).…”

Section: Methodsmentioning

confidence: 99%

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis

Wang

et al. 2016

Journal of Biological Chemistry

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During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of the oocyte is silenced at the fully grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in the oocyte. Recently, RNA methylation has been found to exist in various systems. In this study, we sequenced the N 6 -methyladenosine (m 6 A) modified mRNAs in fully grown germinal vesiclestage and metaphase II-stage oocytes. As a result, we identified 4207 mRNAs with m 6 A peaks in germinal vesicle-stage or metaphase II-stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with protein phosphorylation. Our results suggest that oocyte mRNA methylation can regulate cellular translation and cell division during oocyte meiotic maturation and early embryo development.During oogenesis in animals like Xenopus laevis, mouse, and human, germinal vesicle (GV)-stage 3 oocytes gradually achieve maximum size, and genomic transcription activity is silenced (1, 2). After that, the fully grown GV oocytes resume meiosis and develop to metaphase of the second meiosis (MII) stage. Then the oocyte is fertilized by sperm to form a zygote. The zygote starts mitosis, and embryo development is initiated. As embryo genomic transcription is not reactivated until embryo development to the mid-blastula stage for X. laevis (3) or the 2-cell to 16-cell stages for mammals (4 -7), oocytes or embryos need the maternal RNAs accumulated during oocyte growth to support new protein synthesis.As the synthesis of new proteins such as cyclins is of importance for the meiotic and mitotic events, the translation of maternal mRNAs during oocyte meiotic maturation and early embryo development should be precisely controlled. Previous data have shown that there are mainly two methods for cells to control the maternal mRNA translation in oocyte or early embryo protein binding to the cytoplasmic polyadenylation elements (CPEs) in maternal mRNAs and controlling the polyadenylation of the mRNA poly(A) tails (8). In most cases, shortened poly(A) tails of mRNAs repress their translation, whereas elongated poly(A) tails activate translation (9). The poly(A) tail length of oocyte mRNA is mainly associated with cytoplasmic polyadenylation, which is controlled by RNA binding proteins and associated proteins. The maternal mRNAs, whose 3Ј UTR contains a cis-element CPE, could be bonded by CPE binding...

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Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Cited by 529 publications

References 31 publications

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m ⁶ A-demethylation of NANOG mRNA

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis

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Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Cited by 529 publications

References 31 publications

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

The dual role of N6‐methyladenosine modification of RNAs is involved in human cancers

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m 6 A-demethylation of NANOG mRNA

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis

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Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m ⁶ A-demethylation of NANOG mRNA