Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation.

A bioinformatics pipeline guided by genomic hints of where to look led to the identification and validation of several new classes of self-cleaving ribozymes and several catalytic RNA motifs related to the known hammerhead or HDV ribozymes. Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evidenced by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein we exploited this association to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. We identified three new self-cleaving classes, which we named twister sister, pistol and hatchet, from this collection, suggesting that even more ribozymes remain hidden in modern cells.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Significance: The cyclic dinucleotides c-di-GMP and c-di-AMP are responsible for controlling broad changes in cell phenotypes during the life cycles of many bacterial species. To date, riboswitches that sense c-di-GMP and c-di-AMP have been discovered. The genomic locations of riboswitches reveal numerous genes that are controlled by these signaling compounds and thereby reveal the biological processes that are regulated. Herein, we report that a subset of conserved noncoding RNA domains previously annotated as c-di-GMP-I riboswitches in fact bind the newly discovered second messenger c-AMP-GMP. These riboswitches control many genes involved in the utilization of extracellular iron(III) oxide as an electron sink by various Geobacter species. Our findings reveal additional roles for this recently discovered signaling molecule.

Significance: In the 15 y since metabolite-binding riboswitches were first experimentally validated, only 4 examples of riboswitch classes with altered specificity have been confirmed by experiments out of ∼30 distinct structural architectures. In contrast, evolutionary changes in ligand specificity of proteins are routinely reported. To further investigate the propensity for natural adaptation of riboswitch specificity, we developed a structural bioinformatics method to systematically search for variant riboswitches with altered ligand recognition. This search method yielded evidence for altered specificity within five riboswitch classes, including validation of a second riboswitch class that senses 2′-deoxyguanosine.

Small endonucleolytic ribozymes promote the self-cleavage of their own phosphodiester backbone at a specific linkage. The structures of and the reactions catalysed by members of individual families have been studied in great detail in the past decades. In recent years, bioinformatics studies have uncovered a considerable number of new examples of known catalytic RNA motifs. Importantly, entirely novel ribozyme classes were also discovered, for most of which both structural and biochemical information became rapidly available. However, for the majority of the new ribozymes, which are found in the genomes of a variety of species, a biological function remains elusive. Here, we concentrate on the different approaches to find catalytic RNA motifs in sequence databases. We summarize the emerging principles of RNA catalysis as observed for small endonucleolytic ribozymes. Finally, we address the biological functions of those ribozymes, where relevant information is available and common themes on their cellular activities are emerging. We conclude by speculating on the possibility that the identification and characterization of proteins that we hypothesize to be endogenously associated with catalytic RNA might help in answering the ever-present question of the biological function of the growing number of genomically encoded, small endonucleolytic ribozymes.