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.

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.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq.

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5' fragment with a 2',3' cyclic phosphate (2',3' cP) and a 3' fragment with a 5' hydroxyl (5' OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5' OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3' phosphate of the adapter and then ligates it directly to RNAs with 5' OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3' twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5' OH termini from total RNA.

A rare bacterial RNA motif is implicated in the regulation of the purF gene whose encoded enzyme synthesizes phosphoribosylamine.

The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by using comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound 5-phospho-D-ribosylamine (PRA), which is the first committed step in purine biosynthesis. The consensus model for Fibro-purF motif RNAs includes a predicted three-stem junction that carries numerous conserved nucleotide positions within the regions joining the stems. This architecture appears to be of sufficient size and complexity for the formation of the ligand-binding aptamer portion of a riboswitch. In this study, we conducted biochemical analyses of a representative Fibro-purF motif RNA to confirm that the RNA generally folds according to the predicted consensus model. However, due to the instability of PRA, binding of this ligand candidate by the RNA could not be directly assessed. Genetic analyses were used to demonstrate that Fibro-purF motif RNAs regulate gene expression in accordance with predicted PRA concentrations. These findings indicate that Fibro-purF motif RNAs are genetic regulation elements that likely suppress PRA biosynthesis when sufficient levels of this purine precursor are present.

Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa.

Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme - an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches.

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5' untranslated region of mRNA. Rearrangements of the RNA structure in response, e.g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modelling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behaviour in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyse the influence of 5' hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free licence on GitHub (https://github.com/xileF1337/riboswitch_design).

cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cyclic phosphates and 5' hydroxyl ends.

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].

Novel ribozymes: discovery, catalytic mechanisms, and the quest to understand biological function.

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.

Satellite-Like W-Elements: Repetitive, Transcribed, and Putative Mobile Genetic Factors with Potential Roles for Biology and Evolution of Schistosoma mansoni.

A large portion of animal and plant genomes consists of noncoding DNA. This part includes tandemly repeated sequences and gained attention because it offers exciting insights into genome biology. We investigated satellite-DNA elements of the platyhelminth Schistosoma mansoni, a parasite with remarkable biological features. Schistosoma mansoni lives in the vasculature of humans causing schistosomiasis, a disease of worldwide importance. Schistosomes are the only trematodes that have evolved separate sexes, and the sexual maturation of the female depends on constant pairing with the male. The schistosome karyotype comprises eight chromosome pairs, males are homogametic (ZZ) and females are heterogametic (ZW). Part of the repetitive DNA of S. mansoni are W-elements (WEs), originally discovered as female-specific satellite DNAs in the heterochromatic block of the W-chromosome. Based on new genome and transcriptome data, we performed a reanalysis of the W-element families (WEFs). Besides a new classification of 19 WEFs, we provide first evidence for stage-, sex-, pairing-, gonad-, and strain-specific/preferential transcription of WEs as well as their mobile nature, deduced from autosomal copies of full-length and partial WEs. Structural analyses suggested roles as sources of noncoding RNA-like hammerhead ribozymes, for which we obtained functional evidence. Finally, the variable WEF occurrence in different schistosome species revealed remarkable divergence. From these results, we propose that WEs potentially exert enduring influence on the biology of S. mansoni. Their variable occurrence in different strains, isolates, and species suggests that schistosome WEs may represent genetic factors taking effect on variability and evolution of the family Schistosomatidae.

A streamlined protocol for the detection of mRNA-sRNA interactions using AMT-crosslinking in vitro.

Until recently, RNA-RNA interactions were mainly identified by crosslinking RNAs with interacting proteins, RNA proximity ligation and deep sequencing. Recently, AMT-based direct RNA crosslinking was established. Yet, several steps of these procedures are rather inefficient, reducing the output of identified interaction partners. To increase the local concentration of RNA ends, interacting RNAs are often fragmented. However, the resulting 2',3'-cyclic phosphate and 5'-OH ends are not accepted by T4 RNA ligase and have to be converted to 3'-OH and 5'-phosphate ends. Using an artificial mRNA/sRNA pair, we optimized the workflow downstream of the crosslinking reaction in vitro. The use of a tRNA ligase allows direct fusion of 2',3'-cyclic phosphate and 5'-OH RNA ends.