title: Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay.
abstract: 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.
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Sunny
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Watzinger
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Johannes David
authors.initials: JD
authors.lastname: Hartmann
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Kötter
authors.affiliation: Institute of Molecular and Cellular Microbiology Goethe University, Frankfurt am Main, Germany.
authors.forename: Karl-Dieter
authors.initials: KD
authors.lastname: Entian
mesh_terms: D001483:Base Sequence; D056148:Chromatography, Reverse-Phase; D008745:Methylation; D010940:Plant Proteins / Q000378:metabolism*; D017354:Point Mutation; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics* / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000235:genetics* / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
chemical_list: C526101:Mung Bean Nuclease; D010940:Plant Proteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D020537:RNA, Small Nucleolar; C065039:RNA, ribosomal, 25S; D012266:Ribose; D015719:Single-Strand Specific DNA and RNA Endonucleases