Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus

Abstract

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75 degrees C, compared with 11 and 23 sites, respectively, in Escherichia coli. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2' of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83 degrees C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23 degrees C range shown for 2'-O-methyladenosine in 16S rRNA (21% increase) and for 2'-O-methylcytidine (24%) and 2'-O-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.

FIG. 1

Determination of nucleosides from S. solfataricus 16S rRNA (75°C culture) by LC-MS analysis of an enzymatic digest, using UV detection at 260 nm. Nucleoside identities established from chromatographic retention times and mass spectra: 1, ψ; 2, C; 3, U; 4, m⁵C; 5, Cm; 6, G; 7, Um; 8, Gm; 9, m²G; 10, ac⁴C; 11, A; 12, Am; 13, m⁶A; 14, m₂⁶A. Unnumbered peaks were shown by corresponding mass spectra not to represent nucleosides.

FIG. 2

Chromatographic separation of nucleosides from S. solfataricus 23S rRNA (75°C culture) by LC-MS analysis of an enzymatic digest, using UV detection at 260 nm. Nucleoside identities established from chromatographic retention times and mass spectra: 1, ψ; 2, C; 3, U; 4, m⁵C; 5, Cm; 6, G; 7, Um; 8, m³U; 9, Gm; 10, ac⁴C; 12, Am. Substoichiometric impurities: 13(m²G), 14(m⁶A), and 15(m₂⁶A). Unnumbered peaks were shown by corresponding mass spectra not to represent nucleosides.

FIG. 3

Identification of modified nucleosides in 16S rRNA (A to D) and 23S rRNA (E), using selected ion profiles for the protonated molecule (top sections) and protonated base (bottom sections) ions. (A) 5-Methylcytidine; (B) N⁴-acetylcytidine; (C) N²-methylguanosine; (D) N⁶-methyladenosine; (E) 2′-O-methyluridine (15.3 min) and 3-methyluridine (14.8 min). Relative ion abundance values (ordinates) have been normalized for each compound to 100% in panels A to D and to 100% for the most abundant m/z 259 signal in panel E.