Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay

• Published in: PloS one Vol. 11; no. 12; p. e0168873
• Main Authors: Yang, Jun, Sharma, Sunny, Watzinger, Peter, Hartmann, Johannes David, Kötter, Peter, Entian, Karl-Dieter
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.12.2016; Public Library of Science (PLoS)
• Subjects: Baking yeast; Base Sequence; Biology and life sciences; Biopolymers; Biosynthesis; Catalysis; Chemical synthesis; Chromatography; Chromatography, Reverse-Phase; Enzymes; Ethanol; Eukaryotes; Genetic aspects; Heterogeneity; High performance liquid chromatography; Liquid chromatography; Mathematical analysis; Methylation; Molecular biology; Mutants; Nuclease; Physical Sciences; Physiological aspects; Plant Proteins - metabolism; Point Mutation; Protein biosynthesis; Protein synthesis; Proteins; Research and Analysis Methods; Residues; Ribose; Ribose - metabolism; Ribosomal RNA; Ribosomes; Ribosomes - genetics; Ribosomes - metabolism; Ribozymes; RNA, Fungal - genetics; RNA, Fungal - metabolism; RNA, Ribosomal - genetics; RNA, Ribosomal - metabolism; RNA, Ribosomal, 18S - genetics; RNA, Ribosomal, 18S - metabolism; RNA, Small Nucleolar - genetics; RNA, Small Nucleolar - metabolism; rRNA 18S; rRNA 25S; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Single-Strand Specific DNA and RNA Endonucleases - metabolism; snoRNA; Sucrose; tRNA; Yeast; Belgium; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0168873

Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.