Comparative genomics of small RNAs in bacterial genomes

In recent years, various families of small non-coding RNAs (sRNAs) have been discovered by experimental and computational approaches, both in bacterial and eukaryotic genomes. Although most of them await elucidation of their function, it has been reported that some play important roles in gene regulation. Here we carried out comparative genomics analysis of possible sRNAs that are computationally identified in 30 bacterial genomes from gamma- and alpha-proteobacteria and Deinococcus radiodurans. Identified sRNAs are clustered by a complete-linkage clustering method to see conservation among the organisms. On average, sRNAs are found in approximately 30% of intergenic regions of each genome sequence. Of these, 25.7% are conserved among three or more organisms. Approximately 60% of the conserved sRNAs do not locate in orthologous intergenic regions, implying that sRNAs may be shuffled their positions in genomes. The current study implies that sRNAs may be involved in a more extensive range of functions in bacteria.     

Identification of bacterial small non-coding RNAs: experimental approaches

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed.     

Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of ~60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5′- and 3′-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3′-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3′-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5′ or 3′ ends of the mRNAs with perfect complementarity.     

The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAs using suboptimal RNA structures

Non-coding RNAs (ncRNAs) are regulatory molecules encoded in the intergenic or intragenic regions of the genome. In prokaryotes, biocomputational identification of homologs of known ncRNAs in other species often fails due to weakly evolutionarily conserved sequences, structures, synteny and genome localization, except in the case of evolutionarily closely related species. To eliminate results from weak conservation, we focused on RNA structure, which is the most conserved ncRNA property. Analysis of the structure of one of the few well-studied bacterial ncRNAs, 6S RNA, demonstrated that unlike optimal and consensus structures, suboptimal structures are capable of capturing RNA homology even in divergent bacterial species. A computational procedure for the identification of homologous ncRNAs using suboptimal structures was created. The suggested procedure was applied to strongly divergent bacterial species and was capable of identifying homologous ncRNAs.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

Analysis of similarity within 142 pairs of orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae

Patterns of similarity between genomes of related species reflect the distribution of selective constraint within DNA. We analyzed alignments of 142 orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae and found a mosaic pattern with regions of high similarity (phylogenetic footprints) interspersed with non-alignable sequences. Footprints cover ~20% of intergenic regions, often occur in clumps and are rare within 5′ UTRs but common within 3′ UTRs. The footprints have a higher ratio of transitions to transversions than expected at random and a higher GC content than the rest of the intergenic region. The number of footprints and the GC content of footprints within an intergenic region are higher when genes are oriented so that their 5′ ends form the boundaries of the intergenic region. Overall, the patterns and characteristics identified here, along with other comparative and experimental studies, suggest that many footprints have a regulatory function, although other types of function are also possible. These conclusions may be quite general across eukaryotes, and the characteristics of conserved regulatory elements determined from genomic comparisons can be useful in prediction of regulation sites within individual DNA sequences.     

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.     

Discovery of 17 conserved structural RNAs in fungi

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.