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

Abstract

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA In addition to having 5'-methylated cap structures, many eucaryotic mRNAs contain internal methylated bases. N6-methyladenosine (m6A), the major form of modified nucleoside, has been detected in most higher eucaryotic cell mRNAs, in viral mRNAs, and in the virion RNA of retroviruses (2). Methylation of internal adenosines occurs in the nucleus very rapidly after synthesis of the heterogeneous nuclear RNA precursor to mRNA and before the RNA is spliced (9, 28). While the function of this internal methylation is not known, several observations suggest a significant role for m6A in RNA metabolism.Some implications for the function of internal base modifications come from methylation inhibition studies, using either cycloleucine or S-tubercidinylhomocysteine to block internal methylation of RNA. These experiments suggest an involvement of m6A in RNA processing events or in the transport of mRNA from the nucleus to the cytoplasm (6, 13, 35). It is not clear from these studies, however, that the effect of the inhibitors is directly related to the methylation state of the mRNA being examined. Secondary effects of the drugs have not been ruled out. Furthermore, the requirement for internal methylation in mRNA is not absolute, as mature messages without any detectable m6A residues do exist, including globin and histone mRNAs (24,27).In all cases characterized to date, m6A occurs only in the sequences Gm6AC and Am6AC (7,12,31,38). Preliminary localization studies with specific RNAs have determined that the distribution of m6A within a molecule is nonrandom.Rous sarcoma virus (RSV), for example, has an average of 10 to 15 m6A residues per virion RNA molecule (4, 14, 36).These methylated bases have been detected only in the 3' half of the genomic RNA (4), even though sequence analysis shows that putative methylation sites (GAC and AAC) are widely dispersed throughout the molecule (32). 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.(SV40) late mRNAs, on the other hand, each contain an average of three m6As, all near the 5' ends of those messages in translated sequences (7). Adenovirus mRNAs are methylated in the 5' two-thirds of the molecules, and m6As seem, in this system, to be conserved during mRNA processing (9, 33).We have developed a method for directly identifying m6A residues within the genomic RNA of RSV. We have precisely mapped seven such sites in the genome, all within protein coding regions. In addition, we have analyzed the extent of modification at individual methylation sites. Earlier studies with SV40 (7) and with RSV (12) suggest that methylation at some specific sites is heterogeneous, with bas...

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4.

“…Our results suggest a turnover of late cytoplasmic methylations in hepatocytes, which is selective for exposed 2'-O-ribose moieties. In this regard, the late methylation in mRNA caps occurs in the cytoplasm and involves a 2'-O-methylation (30,42), although internal merA methylations may participate in processing reactions (14). Furthermore, our data suggest the functional importance of the methylation, because 2'-O-methylation of undermethylated ribosomal subunits largely restores the functional deficit.…”

m6a RNA Methylation: The Implications for Health and Disease

“…Around 30% of genes contain microRNA (miRNA) binding sites in the 3'UTRs [3]. Interestingly, where these occur simultaneously the m 6 A modification is found closer to the stop codon, whereas the miRNA binding site appears to be at the opposite end [2]. Without further information this proximity may well be circumstantial and irrelevant, however the occurrence of both together is higher than would be expected by chance alone.…”

“…Not only is this modification found extensively in mRNAs it is also found in non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) [2]. The m 6 A modification has been demonstrated to be an internal modification predominantly found in mature mRNA transcripts [2]. Analysis of these transcripts revealed m 6 A modifications on mRNA transcripts which encoded genes involved in transcriptional regulation, RNA metabolism and signalling cascades [2].…”

“…The m 6 A modification has been demonstrated to be an internal modification predominantly found in mature mRNA transcripts [2]. Analysis of these transcripts revealed m 6 A modifications on mRNA transcripts which encoded genes involved in transcriptional regulation, RNA metabolism and signalling cascades [2]. The number of 3' UTRs of mRNA transcripts demonstrating m 6 A methylation appear to be above average at two thirds, it should be noted however that accuracy of site detection and methylation state is likely to be more accurate for highly expressed genes than genes with low copy numbers.…”

“…Until recently this field had focussed on changes in the chemistry of DNA and the actions of histone proteins and their subsequent modifications. A few key discoveries are set to add a whole new RNA dimension to this exciting field of research, RNA methylation has entered the fray, specifically N-methyl-6-Adenosine -also known as m 6 A or N6-methyladenosine. Although this is not the only RNA modification, over 100 have been identified; it is by far the most abundant [1].…”