The methylated constituents of L cell messenger RNA: Evidence for an unusual cluster at the 5′ terminus

Abstract

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

“…Although the m 6 A mark on RNA was reported many years ago, [1][2][3] A in mRNA affects mRNA splicing, trafficking, translation, and stability; thus, the reversible m 6 A modification in mRNA plays broad and critical roles in fundamental biological processes and disease development. 5,30 METTL3 and METTL14 methyltransferase activities have been examined using mass spectrometry-based assays, which indicated that they form a stable heterodimer that is more active than each component individually.…”

“…RNA methylation at the N 6 position on adenine (m 6 A) was originally identified in the 1970s, [1][2][3] and was shown to be one of the most abundant internal mRNA modifications. 4 These initial reports showed that approximately 0.1% to 0.4% of the adenosines in cellular RNA are m 6 A modified.…”

Sprouts of RNA epigenetics

“…Future Directions m 6 A was first identified in mammalian mRNA in the 1970s [23][24][25] and it is the most abundant internal mRNA modification in oxidative demethylation of m 6 A, 18 these intermediates were not detected in the ALKBH5-catalyzed m 6 A demethylation process. hm 6 A and f 6 A have half-lives of ~3 h under physiological conditions at 37°C, and will be ultimately converted to unmodified A as the product of the FTO-mediated oxidative demethylation.…”

Recent Advancement in Methodology for Understanding Epigenetic Modifications

“…Other site of modification includes the oxygen of the 2'OH moiety, the carbon atom at position 2 and in adenosine, the carbon atoms at position 5 in pyrimidines [17]. The most abundant and common RNA modifications in some viruses and most eukaryotes is N6-methyladenosine (m6A) which accounts for more than 80% of total RNA methylation [18][19][20][21][22][23]. In addition, it is also found in rRNA, tRNA, and small nuclear RNA (snRNA) as well as various long non-coding RNA, such as Xist [24,25].…”