Abstract
We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.
Highlights
In the methylation reaction, a cellular enzyme utilizes S adenosylmethionine as methyl donor mtoodify the heterogeneous nuclear RNA precursor to mRNA before the RNA is spliced (Chen-Kiang et al, 1979)
Potential m6A sites (GAC and AAC) are methylated in any given RNA (Beemon and Keith, 1977; Canaani et al, 1979;Horowitz et al, 1984; Kane andBeemon, 1985).Beyond the strongly conserved consensus sequences, there appears to be some additional signal(s) forbasemodification which is recognized by the methylase
Site-directed Mutagenesis-Based ontheresults of our saline containing Ca2+/Mg2+and incubated with 50 pg/ml pro- original m6A localizations (Kaneand Beemon,1985), the nase in phosphate-buffered saline containing Ca2+/Mg2+for 5 min to methylated residues at nucleotides 7414 and 7424 on theRSV
Summary
Site-directed Mutagenesis-Based ontheresults of our saline containing Ca2+/Mg2+and incubated with 50 pg/ml pro- original m6A localizations (Kaneand Beemon,1985), the nase in phosphate-buffered saline containing Ca2+/Mg2+for 5 min to methylated residues at nucleotides 7414 and 7424 on theRSV remove virions attached to cell surfaces. The lysate was layered ontoa cushion of 5.7 M cesium chloride in 0.1 M EDTA in 12-ml Beckman genome (numbering according to Schwartz etal., 1983) were chosen for the initial mutagenesis studies These two bases make UP one cluster ofm6A sites within the RSV src gene and alsoreside upstream of a potential splice acceptor site at quick seal tubes, and samples were centrifuged for 16-20 h at 35,000 nucleotide 7456, as discussed previously Nuclei were resuspended in 1 ml of lysis buffer, Dounce homogenized 20 times and pelleted again, and this supernatant was added to the cytoplasmic genome andcontains nucleotides 7414 and 7424. Containing 0.5% Nonidet P-40 0.1% sodium deoxycholate, homog- 7434 (31-mer) was synthesized with two nucleotide changes enized five times, and pelleted a final time The supernatant from this washwas discarded; nuclei were resuspended in guanidinium solution. Template DNA was constructed by inserting a 604nucleotide XhoI-Sau3A fragment from pATV8K
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