Context effects on N6-adenosine methylation sites in prolactin mRNA.
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.
Narayan
P
P
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106.
Ludwiczak
R L
RL
Goodwin
E C
EC
Rottman
F M
FM
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D016384:Consensus Sequence; D066298:In Vitro Techniques; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011388:Prolactin / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000225:Adenine; D008780:Methyltransferases; D011388:Prolactin; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; C000730567:6-methyladenine