FIG 2.

m⁵C sites are mapped in MLV RNA at single-nucleotide resolution. (A) Mouse 28S rRNA m⁵C site identification by bisulfite sequencing. IGV tracks display reads aligned to the 3′ end of the mouse 28S rRNA sequence with two known m⁵C sites. The m⁵C modification percentage at each site is indicated. (B) IGV alignments of RNA-BisSeq data from two independent replicates of MLV virion RNA showing the clustered sites in the upstream gag region and the env region. Reads were aligned to the bisulfite-converted MLV genome sequence in which all C residues were changed to T. Sites with high levels of unconverted C (in blue) are indicative of m⁵C sites, with the modification fraction shown on the right. The y axis indicates read depth. (C) IGV alignments of RNA-BisSeq data from two independent replicates of cellular mRNA showing the clustered sites in the upstream gag region and the env region of MLV gRNA transcripts. (D) Distribution and stoichiometry of m⁵C sites on the MLV genome from virion samples. The y axis shows the m⁵C fractions. The x axis indicates the genomic location of the m⁵C sites, with a diagram of the viral genome shown at the top. (E) Distribution and stoichiometry of m⁵C sites on the MLV genome from cellular mRNA samples. (F) Validation of the three high-confidence m⁵C sites located upstream of gag carried out by RT-PCR followed by Sanger sequencing of bisulfite-treated samples obtained from virions or infected cells. Blue arrows show the locations of the m⁵C sites.