Fig 6. Precise mapping of the chemical modifications of 25S rRNA.

For the mapping of chemically identical modifications located adjacent to each other on 25S rRNA, tiling set of overlapping fragments, along with snoRNA deletion mutants were used. The mapping strategy for Gm2791 and Gm2793 to their precise location used in the present study is shown here as an example. A) To map Gm2791 and Gm2793 to their precise location, three fragments protected by oligo 2749, 2791, and 2793 were isolated. B) Overlaid chromatograms of these three fragments. Similarly, to validate the mapping of Am2256, Am2289 and Am2281, we used respective snoRNA deletion mutant–snR63 for Am2256, and snR13 for both Am2280 and Am2281. Where loss of snR13 led to approximately two third reduction in Am peak area, loss of snR63 resulted in only one third reduction. Gm2288 peak remained unaltered and was used as an internal control for the quantification analysis.