m⁶A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover

Abstract

Understanding the biologic role of N 6 -methyladenosine (m 6 A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m 6 A in exons but very rarely in introns. The m 6 A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m 6 A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m 6 As in CA-RNA are within 50 nucleotides of 5 ′ or 3 ′ splice sites, and the vast majority of exons harboring m 6 A in wild-type mouse stem cells is spliced the same in cells lacking the major m 6 A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m 6 As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m 6 A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m 6 A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability. Studying nascent RNA synthesis in cultured cells using very brief pulse labeling with radioactive nucleosides allowed a number of advances in understanding premRNA synthesis and processing in the era before rapid nucleic acid sequencing. Examples include polyA addition on pre-mRNA before completion of mRNA processing and cytoplasmic entry (Darnell et al. 1971;Edmonds et al. 1971) and locating the first known boundaries of eukaryotic polymerase II transcription units through studying labeled nascent adenovirus transcripts (Bachenheimer and Darnell 1975;Evans et al. 1977;Weber et al. 1977).These early experiments were joined by a cell fractionation technique originated by Wuarin and Schibler (1994) that uses a 1 M urea solution to liberate a "chromatin" fraction from nuclei. This fraction provides a stringent purification of growing nascent pre-mRNA chains, isolated as a chromatin-associated RNA fraction (referred to as CA-RNA), along with RNA polymerase II plus all nuclear DNA and associated histones. Using specific labeled DNA probes, Wuarin and Schibler (1994) demonstrated removal in liver cell nuclei of some, but not all, introns from two specific nascent pre-mRNAs: a transcription factor pre-mRNA involved in circadian rhythm and the HMG coA reductase pre-mRNA. Recently, Pandya-Jones and Black (2009) adapted this procedure to study the extent and order of intron removal in cultured human carcinoma cell nuclei, again showing that many, but not all, introns are removed in CA-RNA.

Nuclear TARBP2 drives oncogenic dysregulation of RNA splicing and decay

“…Although methylation of the target introns has not been demonstrated, this suggests existence of a more general link between intron methylation, retention, and nuclear decay (Kilchert et al, 2015). Building on this mechanism, we also found that a fraction of TARBP2 is associated with chromatin, and therefore it is plausible that TARBP2 promotes intron methylation co-transcriptionally, consistent with a recent report that m 6 A marks are deposited on nascent pre-mRNA (Ke et al, 2017). We speculate that the mode of TARBP2-dependent post-transcriptional regulation we describe here may be advantageous because it allows for the fast decoupling of expression of the TARBP2 regulon from transcriptionally controlled levels.…”

“…A methylation impacts intron retention and RNA stability Recently, it was shown that RNA methylation can occur co-transcriptionally, and m 6 A marks can be detected in chromatin-associated RNA (Ke et al, 2017). Analysis of these chromatin-associated RNA m 6 A marks (CA-m 6 A) revealed that there is a significant enrichment of TARBP2-bound introns among chromatin-associated methylated introns ( Figure S3A).…”

“…The prevalent internal RNA modification mark N(6)-methyladenosine (m 6 A) has been reported to play a role in regulating most facets of the RNA life cycle, including regulation of pre-mRNA splicing, mRNA stability, and mRNA translation (Lin et al, 2016;Liu et al, 2015;Wang et al, 2015Wang et al, , 2014Xiao et al, 2016;Zhao et al, 2014). Recently, work by us (Alarcón et al, 2015) and others (Ke et al, 2017;Knuckles et al, 2017) has established that m 6 A marks are deposited in the nucleus and are proposed to function in many nuclear regulatory processes, including microRNA and messenger RNA processing. Despite the widespread use of these pathways, the underlying regulatory programs that influence m 6 A deposition patterns across the transcriptome are poorly characterized.…”

“…This result demonstrates that a portion of nuclear TARBP2 is associated with chromatin, which is consistent with a role for TARBP2 in promoting the co-transcriptional m 6 A methylation of RNA. To independently confirm the intron retention analysis of our RNA-seq data, we randomly selected a number of TARBP2-bound introns with known methylation sites at nucleotide resolution (CA-m6A and MeRIP-Seq; Alarcón et al, 2015a;Ke et al, 2017). We then used qRT-PCR to measure TARBP2-dependent relative changes in retention of this set of introns using exon-exon and exon-intron spanning primers.…”

Pre-mRNA processing includes N6 methylation of adenosine residues that are retained in mRNA exons and the fallacy of “RNA epigenetics”

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AbstractBy using a cell fraction technique that separates chromatin associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown (Ke et al 2017) that residues in exons are methylated (m 6 A) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called "epigenetic" demethylation.

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