RNA methylation to form N6-methyladenosine (m6A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression in diverse physiological processes. Transcriptome-wide m6A mapping has revealed the distribution and pattern of m6A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m6A, providing mechanistic links connecting m6A to cellular differentiation, cancer progression and other processes. The effects of m6A on mRNA are mediated by an expanding list of m6A readers and m6A writer-complex components, as well as potential erasers that currently have unclear relevance to m6A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts - in some cases, controversies - regarding our understanding of the mechanisms and functions of m6A readers, writers and erasers.

Reading, writing and erasing mRNA methylation.

N6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

/pdf/virma-mediates-preferential-m6a-mrna-methylation-in-3-utr-504jwhx0m6.pdf

VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation.

Zc3h13 Regulates Nuclear RNA m6A Methylation and Mouse Embryonic Stem Cell Self-Renewal.

A method of identifying microorganisms with reduced adaptability to a particular environment, wherein (1) each microorganism is independent by insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence. Mutating to provide a plurality of microorganisms, or clones of said microorganisms, such that each mutation has a different label sequence; (2) providing storage samples of each mutation produced by step (1), respectively, and providing storage nucleic acids each comprising a unique label sequence from each mutation; (3) introducing a plurality of mutations produced by step (1) into the specific environment such that the microorganisms capable of growing in the specific environment grow in the above environment; (4) recovering the microorganisms or selected portions thereof from the environment and separating nucleic acids from the recovered microorganisms; (5) comparing all label sequences in the nucleic acid isolated in step (4) with the unique label sequences of each of the stored mutations as in step (2); and (6) any label isolated in step (4). Screening for individual mutations that also do not have a sequence.

Identification of genes

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

/pdf/zc3h13-flacc-is-required-for-adenosine-methylation-by-43iwzkneao.pdf

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-adenosine methylation (m6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6 A writer proteins in Arabidopsis thaliana. The components required for m6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/nph.14586

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

The methyltransferase complex (m6A writer), which catalyzes the deposition of N6-methyladenosine (m6A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Here, using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, are discovered as components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA (mRNA adenosine methylase A). Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation and decreased lateral root formation. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ2 is likely the plant equivalent of ZC3H13 (Flacc) of the metazoan m6A-METTL Associated Complex. 

/pdf/two-zinc-finger-proteins-with-functions-in-m6a-writing-247y8zsx.pdf

Two zinc finger proteins with functions in m6A writing interact with HAKAI

/pdf/two-zinc-finger-proteins-with-functions-in-m6a-writing-25gxtynn.pdf

N6‐methyladenosine (m6A) is one of the most abundant modifications determining the fate of RNA. Currently, m6A modification is tightly connected with tumorigenesis and presents novel promise in clinical applications. Regulated cell death (RCD) is a programmed mechanism that plays a complicated role in malignant transition. Regarding the main forms of RCD, aberrant levels of m6A modification have been detected during the progression of apoptosis, autophagy, ferroptosis, necroptosis, and pyroptosis in several diseases. However, few reviews have elucidated the correlation between m6A‐modified RCD and carcinogenesis. In this review, we summarize the regulators of m6A methylation and their functions in carcinogenesis through an overview of m6A‐modified RCD. Additionally, we assume the potential role of m6A modification regulators as novel biomarkers for chemotherapies and precision medicine. Furthermore, we review the controversies and conflicts in m6A explorations and predict future orientations of m6A‐modified RCD for clinical applications.

Potential applications of N6‐methyladenosine modification in the prognosis and treatment of cancers via modulating apoptosis, autophagy, and ferroptosis

Mi Zhang

Papers

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

Two zinc finger proteins with functions in m6A writing interact with HAKAI

Two zinc finger proteins with functions in m6A writing interact with HAKAI

Potential applications of N6‐methyladenosine modification in the prognosis and treatment of cancers via modulating apoptosis, autophagy, and ferroptosis