Mapping of N6-methyladenosine residues in bovine prolactin mRNA.

Abstract

N6-Methyladenosine (m6A) residues, which are found internally in viral and cellular mRNA populations at the sequences Apm6ApC and Gpm6ApC, have been proposed to play a role in mRNA processing and transport. We have developed a sensitive approach to analyze the level and location of m6A in specific purified cellular mRNAsin an attempt to correlate m6A location with function. Polyadenylylated mRNA is hybridized to cDNA clones representing the full size mRNA under study or fragments of it, and the protected RNA is digested andlabeled with polynucleotide kinase in vitro. After enrichment for m6A with anti-m6A antibody, the [32P]-pm6A is separated on TLC plates, and compared with the total amount of radiolabeled nucleotides. Using this combination of in vitro RNA labeling and antibody selection, we were able to detect m'A in purified stable mRNAs that cannot be readily labeled in cells with greater sensitivity than was possible by previous techniques. We applied this technique to bovine prolactin mRNA and showed that this mRNA contains m6A. Moreoverj all of the m'A residues in this message are found within the 3' two-thirds of the molecule and are highly concentrated (61%) within a sequence of 108 nucleotides at the 3' noncoding region of the message. The nonrandom distribution of m6A in a specific cellular mRNA, as demonstrated for bovine prolactin, will have to be taken into account when designing a model for m6A function.The most prevalent internal methylated nucleoside in eukaryotic mRNA is N6-methyladenosine (m6A). This modified nucleoside is found in RNAs of higher eukaryotic organisms (1-6), plants (7-9), and viruses (3,(10)(11)(12), and occurs at two specific sequences: . The high degree of sequence specificity, together with the fact that m6A is conserved during processing of the heterogeneous nuclear RNA in the nucleus (11,16,18,19), argues for an important biological function. In viral RNA, internal m6A is distributed in a nonrandom fashion. In Rous sarcoma virus, these methylated nucleosides are concentrated in a 3500-nucleotide region of the 10,000-nucleotide RNA genome (12). The highest level of internal m6A residues in simian virus 40 (SV40) nuclear RNA is found in RNA transcripts from two specific regions of the viral genome (20).The m6A-containing oligonucleotides of late SV40 16S and 19S mRNAs were further localized op the viral genome (15). The specificity of m6A location in particular regions of viral RNA implies that, apart from sequence specificity, other features of the RNA may influence the location of internal methylation and is suggestive of a biological role for m6A.Further indication of the role of m6A was obtained by inhibition studies. Inhibition of internal methylation of mRNA by cycloleucine in B77 avian sarcoma virus results in accumulation of genome-length RNA and decrease in the levels of spliced viral mRNA (21). Cycloleucine treatment of SV40-infected BSC-1 cells results in reduction of the amount of m6A residues as well as inhibition of SV40 mRNA production, but almost no ...

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

“…1 and reference 4). In other systems there are differing results on the overall distribution of m6A within RNA (1,7,17). In addition to the minimal recognition sequences, GAC and AAC, some higher order of specificity is needed to explain this varied positioning of m6A in different RNAs.…”

“…Likewise, internal methylation of bovine prolactin mRNA is confined to the 3' two-thirds of the message, with a high concentration of m6A in the 3' noncoding region (17). Simian virus 40 * Corresponding author.…”

Chemical Modifications Mark Alternatively Spliced and Uncapped Messenger RNAs in Arabidopsis

“…While modifications are best characterized in noncoding tRNAs and rRNAs, mRNAs have also been found to contain N 6 -methyladenosine (m 6 A) (Horowitz et al, 1984;Dominissini et al, 2012;Meyer et al, 2012), 5-methylcytosine (m 5 C) (Squires et al, 2012), inosine (I) (Li et al, 2009;Wulff et al, 2011), and pseudouridine (Y) (Carlile et al, 2014;Schwartz et al, 2014b). Additionally, there is a growing body of evidence to support the functional significance of RNA modifications within mRNAs.…”

m6a RNA Methylation: The Implications for Health and Disease

“…Despite its discovery in the 1970s [24], the m 6 A RNA modification has been mostly neglected until recent times, with research focussing on its occurrence [17] and sequence specificity [16,20] rather than its function.…”

“…It is also likely that other binding proteins may enhance or repress binding to the recognition sequence or overlapping sites, for example FTO binding sites [15], thus determining which sites are methylation or non-methylated. The recognition sequence for the m 6 A methylation site is significantly different from other RNA modifications, this sequence has been identified as RRACH (where R = G or A and H = A, C or U) [7,[16][17][18][19][20]. Its occurrence has been estimated at 1 in 2000 bases in humans [2,5].…”