Fig 4. Specific mapping of the chemical modifications of 18S rRNA.

To map the chemical modifications with a single nucleotide resolution, tiling set of overlapping fragments, along with snoRNA, and rDNA point mutants were used. To exemplify the tiling set strategy, Am541 mapping used in the present study is shown. A) To map Am 541 to its precise location, three fragments protected by oligo 491, 527, and 542 were isolated. B) Overlaid chromatograms of these three fragments. To validate the precise location of m⁷G1575, rDNA point mutant was generated where G1575 was exchanged with A in a plasmid-borne copy of 35S rDNA transcribed under the native promoter in a strain where the genomic rDNA was deleted. Exchange of G1575 to A led to complete loss of m⁷G derived from 18S rRNA. As a control, we also used ∆trm112 mutant. Loss of trm112 leads to a complete loss of m⁷G1575 [30]. C) Overlaid chromatograms of isogenic Wild type (WT), G1575A rDNA point, and ∆trm112 mutant. To validate partial modifications at Am100 of 18S rRNA, corresponding snoRNA snR51 was deleted and its contribution to the total Am peak of 18S rRNA was assessed. D) Overlaid chromatograms of isogenic WT and ∆snr51.