Fig 1.
Reversed Phase High Performance Liquid Chromatography (RP-HPLC) analysis of 18S and 25S rRNA of S. cerevisiae (budding yeast).
18S and 25S rRNA of yeast were isolated by sucrose gradient centrifugation. A) Sucrose gradient sedimentation profile of yeast total RNA separated on 5% to 25% sucrose gradient. Fractions corresponding to tRNA (predominantly), 18S rRNA, and 25S rRNA were collected, and the rRNAs were isolated using 95% ethanol precipitation at -80°C. B) 1.5% Agarose gel showing the rRNA recovered after ethanol precipitation. Yeast total RNA was used as a control. Both 18S and 25S rRNA were digested to nucleosides using P1 nuclease and alkaline phosphatase. Nucleosides derived from 18S rRNA and 25S rRNA were analyzed by RP-HPLC. C) RP-HPLC chromatogram of 18S rRNA, and D) 25S rRNA, showing peaks corresponding to major nucleosides labeled in black and of modified residues in red.
Table 1.
RP-HPLC quantification of Nucleosides in S.cerevisiae 18S and 25S rRNA.
Fig 2.
Mung bean nuclease protection assay.
A) Schematic illustration of MBN protection assay used in the present study for the analysis of chemical modifications of 18S rRNA, and B) 25S rRNA. 45 distinct fragments from 18S, and 68 separate fragments from 25S rRNA were isolated for mapping of their chemical constituents. C) Graphical illustration for the use of tiling set of overlapping fragments used in the present study to map the modified residues to a single nucleotide resolution. The fragments protected from MBN were isolated from the debris by 7 M Urea 13% PAGE gel. D) Representative gel for the MBN assay showing an intact protected fragment retrieved after the MBN digestion. The synthetic antisense oligonucleotide used for the protection is used as a marker (50 nts). All fragments isolated from 18S and 25S rRNA were extracted from the gel and digested to nucleosides for their composition analysis.
Fig 3.
Identification and mapping of complete set of ribose and base modifications of 18S rRNA.
Analysis of the composition of 45 fragments derived from 18S rRNA using MBN assay permitted identification of all ribose and base modifications of 18S rRNA. A) Overlaid chromatograms of different fragments identified to contain Am residues; (B) Gm residues; (C) Cm residues; (D) Um residues; (E) m7G, and (F) ac4C residues. Location of each modification along with the oligonucleotide number used for the isolation of respective fragment are mentioned on the right side of the peak.
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.
Table 2.
Approximate values for the extent of modifications in 18S and 25S rRNA, using RP-HPLC and its comparison with RiboMethSeq and SILNAS analyses.
Fig 5.
Identification and mapping of complete set of ribose and base modifications of 25S rRNA.
Composition analysis of the 68 discrete fragments isolated from 25S rRNA using MBN protection assay. Overlaid RP-HPLC chromatograms of different fragments identified to contain (A) Am residues; (B) Gm residues; (C) Cm residues; (D) Um residues; (E) m1A; (F) m5C; and (G) m3U residues. Location of each modification along with the oligonucleotide number used for the isolation of respective fragment are stated on the right side of the peak. For the fragments containing more than one modifications, e.g. oligo 45, oligo 46 for Am, oligo 18 and 56 for Gm, and oligo 49 and 55 for Um, strategy explained above in Fig 2C were used to map them to exact position.
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.
Fig 7.
3D and 2D modification atlas for the chemical modifications of yeast rRNA.
Both ribose and base modifications analyzed in the present study are mapped on the 3D structure of ribosome. 3D cartoon of the yeast 18S rRNA (A) and 25S rRNA (B), highlighting the location of ribose methylations (purple spheres), and base modifications (orange spheres). PDB files 3U5B and 3U5D were used for the representation of 18S and 25S ribosomal RNA. The cartoon was made by PyMOL software (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.). Both ribose and base modifications were also mapped on to the 2D sequence map of the 18S (C) and 25S (D) rRNA of the yeast using online RiboVision suite (http://apollo.chemistry.gatech.edu/RiboVision/).