Discovery of natural non-circular permutations in non-coding RNAs.

Research in the last two decades has increasingly demonstrated that RNA has capabilities comparable to those of proteins, for example the ability to form intricate 3D structures necessary for catalysis. Numerous protein domains are known in varied within-domain rearrangements, called permutations, that change the N- to C-terminal order of important amino acids inside the domain, but maintain their 3D locations. In RNAs, by contrast, only simple circular permutations are known, in which 5' and 3' portions of the molecule are merely swapped. Here, we computationally find and experimentally validate naturally occurring RNAs exhibiting non-circular permutations of previously established hammerhead ribozyme RNAs. In addition to the rearranged RNAs, a bioinformatics-based search uncovered many other new conserved RNA structures that likely play different biological roles. Our results further demonstrate the structural sophistication of RNA, indicate a need for more nuance in the analysis of pseudoknots, and could be exploited in RNA-based biotechnology applications.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Identification of over 200-fold more hairpin ribozymes than previously known in diverse circular RNAs.

Self-cleaving ribozymes are catalytic RNAs that cut themselves at a specific inter-nucleotide linkage. They serve as a model of RNA catalysis, and as an important tool in biotechnology. For most of the nine known structural classes of self-cleaving ribozymes, at least hundreds of examples are known, and some are present in multiple domains of life. By contrast, only four unique examples of the hairpin ribozyme class are known, despite its discovery in 1986. We bioinformatically predicted 941 unique hairpin ribozymes of a different permuted form from the four previously known hairpin ribozymes, and experimentally confirmed several diverse predictions. These results profoundly expand the number of natural hairpin ribozymes, enabling biochemical analysis based on natural sequences, and suggest that a distinct permuted form is more biologically relevant. Moreover, all novel hairpins were discovered in metatranscriptomes. They apparently reside in RNA molecules that vary both in size-from 381 to 5170 nucleotides-and in protein content. The RNA molecules likely replicate as circular single-stranded RNAs, and potentially provide a dramatic increase in diversity of such RNAs. Moreover, these organisms have eluded previous attempts to isolate RNA viruses from metatranscriptomes-suggesting a significant untapped universe of viruses or other organisms hidden within metatranscriptome sequences.

Discovery and characterization of a fourth class of guanidine riboswitches.

Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.

Discovery of 20 novel ribosomal leader candidates in bacteria and archaea.

BACKGROUND: RNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5' UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted. RESULTS: By analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites. CONCLUSIONS: This work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.