Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes

Abstract

Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding 'intergenic regions' (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel riboswitch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.

Discovering riboswitches: the past and the future

“…Intriguingly, there is a large collection of 'orphan' riboswitch candidates (Box 3) that are known to exhibit some characteristics consistent with riboswitch function, but whose ligands remain to be established [138][139][140]171]. If the longest-standing orphan riboswitch candidates [171] are assumed to represent novel riboswitch classes and are added to the power law graph (Figure I, right), they extend the linear portion of the data without substantially changing the key parameters derived from the power law equation, as noted previously.…”

“…Many of these RNAs have resisted efforts to identify their target ligands (Figure I), and are thus considered to be 'orphan' riboswitch candidates [37]. At this time, ~100 candidate orphan riboswitch classes await validation [37,[138][139][140]171], each presenting challenges for those who wish to embark on riboswitch validation studies.…”

“…Bioinformatic searches Most of the methods described previously could yield novel riboswitch class discoveries, but they are not the high-probability, scalable methods for the discovery of novel riboswitch classes that will substantially advance the field. By contrast, bioinformatic methods [37,[137][138][139][140][141][142][143] can be applied to entire genomic databases, and they can be indifferent to the riboswitch class or the identities of the ligands sensed. These search algorithms exploit a comparative sequence analysis approach [144,145] to reveal novel RNA motifs with conserved sequence and structure features [146][147][148].…”

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2′-deoxyguanosine

“…Even with these restrictions, metabolite-binding riboswitches demonstrate a remarkable ability to recognize a wide range of biological ligands, such as signaling molecules, elemental ions, coenzymes, amino acids, and nucleotide derivatives ( 7 ). To date, more than 50 different riboswitch classes have been experimentally validated and more than 100 additional “orphan” ( 8 ) riboswitch candidates have been reported ( 9 – 11 ). Furthermore, it has been proposed that many hundreds of riboswitch classes have yet to be discovered just among the bacteria whose genomes have already been sequenced ( 7 , 12 , 13 ).…”

Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products

“…Its HMP‐PP (thiamin precursor) binding aptamer is embedded in an intrinsic transcription terminator, and ligand binding relieves transcription termination (Atilho et al, 2019). Novel riboswitches are discovered by comparative sequence analyses (Brewer, Greenlee, et al, 2021).…”