Abstract
The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells.
Highlights
Localization of six new NB-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12)
GAC and AAC should appear every 32 nucleotides in a random sequence; only one m6A residue is seen per approximately 1000 nucleotides in total mammalian cell mRNA (Adams and Cory, 1975; Desrosiers et al, 1975; Lavi et al, 1977; Perry et al, 1975; Wei et al, 1976)
0.14%, using in uiuo 32P-labeled RNA. This suggested an average of 12.6 m6Ap residues/9.5-kilobase RNA, in good agreement with the level previously observed for Prague B RSV RNA (Beemon and Keith, 1977)
Summary
Localization of six new NB-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA Inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity.
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