Identification of a large noncoding RNA in extremophilic eubacteria

Abstract

We have discovered a large and highly conserved RNA motif that typically resides in a noncoding section of a multigene messenger RNA in extremophilic Gram-positive eubacteria. RNAs of this class adopt an ornate secondary structure, are large compared with most other noncoding RNAs, and have been identified only in certain extremophilic bacteria. These ornate, large, extremophilic (OLE) RNAs have a length of approximately 610 nucleotides, and the 35 representatives examined exhibit extraordinary conservation of nucleotide sequence and base pairing. Structural probing of the OLE RNA from Bacillus halodurans corroborates a complex secondary structure model predicted from comparative sequence analysis. The patterns of structural conservation, and its unique phylogenetic distribution, suggest that OLE RNA carries out a complex and critical function only in certain extremophilic bacteria.

Fig. 1.

Sequence alignment and predicted base-pairing interactions of 15 OLE RNA representatives. Nucleotides shaded in the same color identify stretches of complementary base pairs labeled P1 through P14. Black and gray labels designate 5′ and 3′ portions of the base-paired elements, respectively. The “Consensus” line was determined by including 20 OLE RNA representatives isolated by PCR amplification of environmental sequences [see supporting information (SI) Fig. 5 for a complete alignment]. Nucleotides that exhibit 97% or greater conservation (red) or that exhibit 75% or greater conservation (black) are depicted. R is G or A; Y is C or U; M is A or C; W is A or U; S is C or G; K is G or U; H is A, C, or U; D is A, G, or U; B is C, G, or U.

Fig. 2.

Conserved nucleotide sequence and secondary structure model for OLE RNAs. Each circle represents a nucleotide whose base identity is not conserved, and thick lines represent regions of variable nucleotide number and identity. Dashed lines indicate continuations of the RNA chain. Three of the 35 representatives carry a short hairpin loop in place of P9.3 (boxed). Other details are as defined in Fig. 1 legend.

Fig. 3.

Expression of OLE RNA in B. halodurans. (A) Schematic representation of the OLE RNA gene and its surroundings in B. halodurans. Box identifies genes present in the same transcriptional unit. Arrows represent the primers used for RT-PCR, where primer pairs are represented as X and X′. Gene designations are as follows: a, BH2786, hypothetical protein; b, nusB, transcriptional antiterminator; c, folD, methylenetetrahydrofolate dehydrogenase; d, BH2783, exodeoxyribonuclease VII (large subunit); e, BH2782, exodeoxyribonuclease VII (small subunit); f, BH2781, geranyltranstransferase; g, BH2780, hypothetical protein; h, dxs, 1-deoxyxylulose-5-phosphate synthase; i, BH2778, hemolysin-like protein; j, ahrC, arginine repressor; k, recN, DNA repair and genetic recombination; l, spoIVB, intercompartmental signaling of pro-σ^K processing/activation. (B) Products of RT-PCR and PCR assays for various primer pairs separated by 1% agarose gel and stained with ethidium bromide. Lanes designated by + and − were PCR-amplified with or without reverse transcription, respectively. M, lanes with DNA markers at 100-bp size increments. (C) 5′ RACE mapping of termini produced by processing of OLE RNAs. Multiple asterisks reflect the number of clones examined that are terminated at the nucleotide indicated.

Fig. 4.

OLE RNA is large and complex, and has an unusual phylogenetic distribution compared with other noncoding RNAs that are widespread in bacteria. (A) Sizes and functions of some noncoding RNAs in B. halodurans. Bars represent size ranges when multiple representatives of an RNA class are known. The size of the group II ribozyme without its internal ORF is presented. Although tmRNAs are translated, their complex structure and multiple functions were reasons for comparison with noncoding RNAs. Note that 16S (≈1,500 nt) and 23S (≈2,900 nt) rRNAs (shaded) are too large for the length scale. (B) Some characteristics of bacteria that carry OLE RNA. Identities of bacterial species 1–15 are the organisms in the same order as they appear in Fig. 1. Solvent tolerance was assigned to those organisms that are known to use or produce high concentrations of organic solvents such as ethanol or acetate. All organisms listed are able to grow under anoxic conditions, although some organisms (e.g., B. halodurans) are facultative anaerobes. (C) The P12.2 through P14.1 region of OLE RNAs exhibits dual symmetry in conserved sequence and secondary structure as indicated by colored shading and boxes.