2′- O -Methylation of Adenosine, Guanosine, Uridine, and Cytidine in RNA of Isolated Rat Liver Nuclei

Abstract

Nuclei isolated from rat liver, when incubated with methyl-labeled S-adenosylmethionine, incorporated label into 2'-O-methyladenosine, 2'-0-methylguanosine, 2'-O-methyluridine, and 2'-0-methylcytidine of endogenous nuclear RNA. Addition of ribosomal RNA rrom wheat germ to the reaction markedly stimulated 2'-O-methylation of total RNA. The relative incorporation of label into the four different 2'-O-methyl ribonucleosides was greatly chahged by the addition of wheat germ RNA. There was much more 2'-O-methylation of the purine ribonucleosides, relative to the pyrimidine ribonucleosides, in the reaction stimulated by wheat germ RNA.Newly synthesized RNA is enzymatically modified in mam-:malian cell nuclei before it is able to function in its cytoplasmic role in protein synthesis (1-3). One way in which newly synthesized RNA is "processed" is the insertion of methyl groups on the 2'-oxygen atom of ribose (sugarmethylation) of all four-of the RNA nucleosides, to yield nucleoside 2'--methyl ethers (4). Although isolated mammalian nucleolar preparations have been reported to contain such sugar-methylating activity (5, 6), the products of sugarmhethylation of RNA; namely Am, Gm, Um, and Cm, have not been conclusively identified. We have recently developed a simple paper chromatographic method for the separation of the four commonly occurring sugar-methylated ribonucleosides from each other, as well as from all of the commonly occurring base-methylated ribonucleosides (7). Using this method, we now report, for the first time, that an enzyme system found in isolated rat liver nuclei will transfer a methyl group from S-adenosyl methionine to the 2'-oxygen of A, G, U, and C in endogenous nuclear RNA. Moreover, we have found that the addition of wheat germ RNA to the reaction mixture greatly stimulates the transfer of methyl groups from S-adenosyl methionine to the 2'-oxygen of RNA (Fig. 1) with 100 ml of 0.085 M Tris-formate, pH 7.8-7 M urea (8). The dinucleotides were then eluted as a second fraction (Fig. 1) t. The molarity of all Tris-formate buffers is expressed in terms of the Tris, rather than formate, concentration.

Reviving the RNA World: An Insight into the Appearance of RNA Methyltransferases

“…Ribose 2′- O -methylation occurs in rRNA, tRNA, mRNA, snoRNA, and siRNA etc. at adenosine, guanosine, cytidine, and uridine nucleobases ( Al-Arif and Sporn, 1972 ) and is ubiquitous in viruses, archaebacteria, eubacteria, yeasts, protists, fungi, and higher eukaryotes ( Feder et al, 2003 ). 2′- O -methylation of RNA by other RNA molecules (ribozyme) or RNA complexes is indeed the primordial manifestation of the methyl transfer reaction and is the most likely mechanistic platform for the de novo creation of deoxyribose sugar (and, in fact, the creation of the DNA molecule) on Earth before the appearance of genetic code or ribonucleotide reductase.…”

RNA methylation by Radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift

“…1). Commonly, RNA methylation is achieved by addition of substrate nucleophile (either a heteroatom or an enzyme-bound enolate) into the electrophylic methyl group of S-adenosyl-L-methionine (SAM) (11)(12)(13). By their reactivity and electronic demands, RlmN and Cfr substrate sites are distinct from other known methylation substrates in RNA, implying a unique mechanism of methylation.…”

Selecting Molecular Recognition. What Can Existing Aptamers Tell Us about Their Inherent Recognition Capabilities and Modes of Interaction?

“…In retrospect, the relative stability of O -Methyl modified RNA has to be viewed in the context of its natural occurrence. 2'- O -methyl modifications of RNA were found early (1966) in E. coli [24], and the specific methylation products were later identified in rat liver RNA (1972) [25]. Interestingly, the dominant substrate of enzymatic 2'-methylation in these studies of alkaline resistant RNA turned out to be purines.…”