Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans

Abstract

The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified.

FIGURE 1.

Ψ sequencing analysis of 23S RNA from D. radiodurans (A) and H. marismortui (B). RNA was treated with (+) or without (−) CMC followed by treatment with alkali (hr OH⁻) for 4 or 6 h, and then used as template for primer extension with reverse transcriptase (Ofengand et al. 2001a). In this assay, a CMC-dependent reverse transcription stop one base 3′ of a uridine (indicated by arrows) identifies a Ψ. The position of each Ψ is numbered and E. coli equivalents are given in parentheses. RNA sequence is shown in A, C, G, and U lanes. (*) Putative m³Ψ. The autoradiographs shown are the only Ψ found in two comprehensive analyses covering residues 1–2852 (99%) of D. radiodurans and residues 1–595, 633–2887 (98%) of H. marismortui 23S RNA. The stop in all lanes in B corresponding to m³U2619 is indicated.

FIGURE 2.

Sequencing mass spectrum of RNase T1 fragment M_r 3550.5 from D. radiodurans LSU RNA showing m³Ψ at position 1898. The spectrum is represented in maximum entropy deconvolution (MaxEnt3) format.

FIGURE 3.

Location of Ψ on secondary structures of 23S RNA from D. radiodurans (A) and H. marismortui (B). In each panel, the secondary structure of the 3′ half of 23S RNA (adapted from the Comparative RNA site, http://www.rna.icmb.utexas.edu) is shown. The regions of the molecule that contain Ψ (bold backbone tracing) are shown in more detail to the right. (*) m³Ψ. Numbering as in Figure 1. The locations of Ψ in other organisms are as indicated. (E) E. coli; (B) B. subtilis; (Z) Z. mays choloroplasts; (A) H. halobium; (S) S. acidocaldarius; (Y) Saccharomyces cerevisiae; (G) Euglena gracilis; (D) D. melanogaster; (M) M. musculus; (H) H. sapiens (Ofengand 2002; Russell et al. 2004; M.W. Gray, pers. commun.).

FIGURE 4.

Ψ sequencing of 23S RNA from E. coli Ψ synthase mutants complemented with putative Ψ synthases from D. radiodurans. (A) The E. coli Ψ synthase mutant ΔrluD transformed with plasmid pTrc99A carrying no insert (p) or RluD from E. coli (pDE) or D. radiodurans (pDD). (B) The E. coli Ψ synthase mutant ΔrluB::kan transformed with plasmid pTrc99A carrying no insert (p) or RluB from E. coli (pBE) or D. radiodurans (pBD). In both panels, Ψ are indicated by E. coli numbering and RNA sequence is shown in A, C, G, and U lanes. Transformation, growth of uninduced transformed cells, and isolation of total RNA was done as described (Del Campo et al. 2001).