Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

Abstract

Identification and characterization of functional elements in the noncoding regions of genomes is an elusive and timeconsuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of noncoding sequence analysis, although they may conceal a wide diversity of novel RNA genes, riboswitches, or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all noncoding elements of a selected reference species in a single pass. This method clusters noncoding elements based on their profile of presence among species. Most noncoding RNAs (ncRNAs) display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphylococcus aureus to experimental validation and confirmed the presence of seven novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path toward the identification of functional relationships between co-evolving coding and noncoding elements.[Supplemental material is available online at www.genome.org.]In all living organisms, the genome regions located between protein-coding sequences are home to a wide diversity of functional elements that include noncoding RNA (ncRNA) genes, DNA regulatory elements, untranslated regions (UTRs) of genes, transposable and self-replicating elements, and a variety of other transcribed or nontranscribed functional sequences. As these elements are often key players in gene regulation and thus in the global cell interaction network, their systematic identification and characterization has become a major challenge in biology.Computational protocols developed to collect and characterize noncoding elements in genomic sequences rely, to a large extent, on comparative genomics. The most common strategies involve, first, collecting sequences under selective pressure and, second, analyzing the aligned sequences using various classifiers that exploit criteria such as nucleotide composition, folding potential, fold conservation, or covariation between distant positions (Rivas and Eddy 2001;Washietl et al. 2005;Pedersen et al. 2006;Torarinsson et al. 2006). In general, such classifiers are designed to detect structured RNAs among noncoding elements with no further distinction between regulatory elements, repeats, or artifacts produced by sequence comparison algorithms.Comparative genomics entails a significant amount of expert intervention, especially in obtaining the right genome set to optimize the specificity and sensitivity of RNA detection. Although a number of studies have successfully identified ncRNAs in several animal (Missal et al. 2005;Washietl et al. 2007) and microbial genomes (Altuvia 2007), the pace at which such studies are performed and published lags far behind the rate of genome sequence output. Most of the complete...

RsaOG, a new Staphylococcal family of highly transcribed non-coding RNA

“…This arrangement suggests that the remaining part of the longer conserved region could be involved in interactions with potential RsaOG targets because such non-structured conserved elements in sRNAs are often involved in trans-pairing. 3 Taken together, these results suggest that the pseudoknot could, by constraining the structure, protect this conserved unpaired region and nucleate or facilitate pairing to the target.…”

“…3 A northern blot experiment indicated that the rsaOG gene was highly expressed. RsaOG was detected at all growth stages but with a progressive increase in post-exponential and stationary phase.…”

“…Recent and ongoing studies are now changing this view. 3,4 We described here a new Staphylococcal RNA, RsaOG, that is likely involved in trans-acting regulatory functions. The fact that RsaOG is expressed during all growth stages suggests a persistent repression or activation function for this gene (or a fine tuning role involving a second factor).…”

“…3 In addition, a microarray study suggests a σ B -dependent regulation of SA2104 and the presence of a σ B -dependent promoter located downstream of RsaOG. 8 These observations strongly support that SA2104 is transcribed independently of rsaOG.…”

“…Known sRNAs in S. aureus include RNAIII 1 and Pathogenicity Island specific ncRNAs. 2 The ncRNA RsaOG (acronym for RNA S. aureus Orsay G) was initially discovered in a bioinformatics ncRNA screen of the S. aureus genome 3 and confirmed in a second study. 4 Here, we show that it is the founding member of a new trans-acting ncRNA family conserved in all Staphylococcaceae.…”