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 Ϸ610 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.isoprenoid ͉ riboswitch ͉ ribozyme ͉ superoperon I n recent years, a large number of small noncoding RNAs (1-3) have been identified in eubacteria (4), archaea (5), and eukaryotes (6). In many instances, novel noncoding RNAs in bacteria have been discovered by using bioinformatics search strategies (e.g., refs. 7-13) that take advantage of the conserved nucleotide sequences and secondary structures of these functional RNAs. Another productive way to identify numerous noncoding RNAs from bacteria is by cloning and sequencing small RNAs isolated from cell extracts (e.g., refs. 13-16), and this approach is useful particularly for those examples that are not well conserved through evolution.RNAs such as tRNAs, rRNAs, and some ribozymes have noncoding functions that have long been known to be central to RNA processing and protein synthesis mechanisms. However, it seems possible that additional noncoding RNAs will be found that perform fundamental biochemical tasks that to date have been assumed to be the exclusive province of protein factors. A number of noncoding RNAs discovered recently have proven to participate as the key components of gene regulation systems (e.g., refs. 17-19). Additional examples of widespread noncoding RNAs in bacteria, such as 6S RNA (20-22), the dual-function tmRNA (23), and noncoding portions of messenger RNAs such as T-Box elements (24) and riboswitches (25-27) perform important gene control and molecular sensing tasks that are critical for cells to function normally. The existence of so many RNAs with atypical functions implies that some newly discovered noncoding RNAs might perform surprising and important roles in fundamental cellular processes. In this article, we describe features of a noncoding RNA element whose size, structural sophistication, and unique phylogenetic distribution are suggestive of a complex biological function. ResultsIdentifying Ornate, Large, Extremophilic (OLE) RNAs by Using Bioinformatics. We have previously used computer-aided search strategies that employ comparative sequence analysis (e.g., refs. 11 and 28-30) to identify noncoding RNAs whose nucleotide sequences and secondary structures are c...

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.

Long 3′ untranslated regions (3′UTRs) are common in eukaryotic mRNAs. In contrast, long 3′UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3′UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3′UTR increases the hilD mRNA level, suggesting that the hilD 3′UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3′UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3′UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3′UTR mRNAs, suggesting that the hilD 3′UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3′UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3′UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3′UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.

Hepatitis C virus (HCV) infection is one of the world's major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: selection for binding and selection for cleaving. The selection protocol was applied to a population of more than 10(15) variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%.

Summary OLE RNAs are large, noncoding transcripts characterized by their ornate secondary structure and presence predominantly in Gram-positive, extremophilic bacteria. A gene for an OLE-associated protein (OAP) is almost always located immediately downstream of the OLE gene. OAP has no extensive homology to other proteins and is predicted to form multiple transmembrane domains. We show that this protein forms a ribonucleoprotein complex with OLE RNA using at least 2:1 protein:RNA stoichiometry. A series of truncated OLE RNA constructs was used to establish that most of the RNA can be deleted without eliminating protein binding. Two primary binding sites are present within the RNA, although additional binding determinants exist and extensive structural stabilization is induced by OAP. RNA fluorescence in situ hybridization was used in Escherichia coli to demonstrate that ribonucleoprotein complex formation localizes the RNA near cell membranes of this heterologous system. Therefore the majority of the complex structure formed by OLE RNA may perform a biochemical function that requires membrane localization.