Context effects on N⁶-adenosine methylation sites in prolactin mRNA

Abstract

The methylation of internal adenosine residues in mRNA only occurs within GAC or AAC sequences. Although both of these sequence motifs are utilized, a general preference has been noted for the extended sequence RGACU. Not all RGACU sequences in an mRNA are methylated and the mechanisms that govern the selection of methylation sites in mRNA remain unclear. To address this problem we have examined the methylation of transcripts containing sequences of a natural mRNA, namely, bovine prolactin mRNA. In this mRNA, a specific AGACU sequence in the 3' untranslated region is the predominant site of methylation both in vivo and in vitro. The degree to which N6-adenosine methyltransferase recognizes the sequence context of the consensus methylation site was explored by mutational analysis of the nucleotides adjacent to the core sequence as well as the extended regions in which the core element was found. Our results indicate that efficient methylation depends on the extended five nucleotide consensus sequence but is strongly influenced by the context in which the consensus sequence occurs within the overall mRNA molecule. Furthermore, consensus methylation sites present in an RNA duplex are not recognized by the methyltransferase.

Post-transcriptional gene regulation by mRNA modifications

“…The amount of m 6 A in isolated RNA is estimated to constitute 0.1–0.4% of all adenosine nucleotides in mammals 18,28 , 0.7–0.9% of adenine nucleotides (all within GA dinucleotides) in meiotic Saccharomyces cerevisiae 29 , and 1–15 sites per virion RNA molecule in various viruses 30 . Mutation and in vitro enzymatic studies have identified a consensus motif of RRm 6 ACH ([G/A/U][G>A]m 6 AC[U>A>C]) 31–34 . However, owing to the low abundance of m 6 A in mRNA and the lack of effective techniques, functional characterizations of m 6 A have been largely absent over the past few decades.…”

RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation

“…Studies in the 1970s revealed that m 6 A modification in mRNA mainly occurs at Pu[G > A]m 6 AC[U > A > C] (Pu represents purine) and is estimated to be present at an average level of approximately three m 6 A residues per mRNA Narayan and Rottman 1988;Csepany et al 1990;Narayan et al 1994). Transcriptome-wide mapping of m 6 A is hindered by the following two facts: (1) m 6 A, akin to A, reverse-transcribes to a thymine (T), and (2) m 6 A is not susceptible to chemical modifications that might promote its detection.…”

The biosynthesis and functional roles of methylated nucleosides in eukaryotic mRNA

“…The only cellular mRNA for which individual m 6 A sites have been mapped is that encoding bovine prolactin (bPRL) (Horowitz et al 1984;Narayan et al 1994). Unlike viral mRNA, labeling steady-state mRNA populations in eukaryotic cells with 32 P-orthophosphate or [ 3 H-methyl]methionine results in the incorporation of low amounts of radioactivity into individual mRNAs.…”

“…A rapid assay for m 6 A formation and the development of an in vitro system for studying m 6 A formation in nuclear extracts facilitated the study of this methyltransferase (Narayan and Rottman 1988;Narayan et al 1994;Bokar et al 1994). Briefly, a synthetic RNA substrate of 60 nucleotides of the 3'-terminus of bovine prolactin mRNA was used as a substrate for methyl transfer from [ 3 Hmethyl]AdoMet.…”