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 5ʹ 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 3ʹ 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 5ʹ OH termini from total RNA.

An RNA Triangle with Six Ribozyme Units Can Promote a Trans-Splicing Reaction through Trimerization of Unit Ribozyme Dimers

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product.

A combinatorial method to isolate short ribozymes from complex ribozyme libraries

In vitro selections are the only known methods to generate catalytic RNAs (ribozymes) that do not exist in nature. Such new ribozymes are used as biochemical tools, or to address questions on early stages of life. In both cases, it is helpful to identify the shortest possible ribozymes since they are easier to deploy as a tool, and because they are more likely to have emerged in a prebiotic environment. One of our previous selection experiments led to a library containing hundreds of different ribozyme clusters that catalyze the triphosphorylation of their 5′-terminus. This selection showed that RNA systems can use the prebiotically plausible molecule cyclic trimetaphosphate as an energy source. From this selected ribozyme library, the shortest ribozyme that was previously identified had a length of 67 nucleotides. Here we describe a combinatorial method to identify short ribozymes from libraries containing many ribozymes. Using this protocol on the library of triphosphorylation ribozymes, we identified a 17-nucleotide sequence motif embedded in a 44-nucleotide pseudoknot structure. The described combinatorial approach can be used to analyze libraries obtained by different in vitro selection experiments.

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes

Ribozymes are catalytic RNAs present in modern genomes but regarded as remnants of a prebiotic RNA world. The paradigmatic hammerhead ribozyme (HHR) is a small self-cleaving motif widespread from bacterial to human genomes. Here, we report that most of the classical type I HHRs frequently found in the genomes of animals are contained within a novel family of non-autonomous non-LTR retrotransposons of the retrozyme class. These retroelements are expressed as abundant linear and circular RNAs of ∼170-400 nt in different animal tissues. Bioinformatic and in vitro analyses indicate an efficient self-cleavage of the HHRs harboured in most invertebrate retrozymes, whereas HHRs in retrozymes of vertebrates, such as the axolotl and other amphibians, require to act as dimeric motifs to reach higher self-cleavage rates. Ligation assays of retrozyme RNAs with a protein ligase versus HHR self-ligation indicate that, most likely, tRNA ligases and not the ribozymes are involved in the step of RNA circularization. Altogether, these results confirm the existence of a new and conserved pathway in animals and, likely, eukaryotes in general, for the efficient biosynthesis of RNA circles through small ribozymes, which opens the door for the development of new tools in the emerging field of study of circRNAs.

Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

Ribozymes are catalytic RNAs present in modern genomes but considered as remnants of a prebiotic RNA world. The paradigmatic hammerhead ribozyme (HHR) is a small self-cleaving motif widespread from bacterial to human genomes. Here, we report that most of the classical type I HHRs frequently found in the genomes of diverse animals are contained within a novel family of non-autonomous non-LTR retrotransposons. These retroelements are expressed as abundant linear and circular RNAs of ~170-400 nt in different animal tissues. In vitro analyses confirm an efficient self-cleavage of the HHRs harboured in invertebrate retrozymes, whereas those in retrozymes of vertebrates, such as the axolotl, require to act as dimeric motifs to reach higher self-cleavage rates. Ligation assays of retrozyme RNAs with a protein ligase versus HHR self-ligation indicate that, most likely, tRNA ligases and not the ribozymes are involved in the step of RNA circularization. Altogether, these results confirm the existence of a new and conserved pathway in animals and, likely, in eukaryotes in general, for the efficient biosynthesis of RNA circles through small ribozymes, which will allow the development of biotechnological tools in the emerging field of circRNAs.

3. RNA

The first RNA molecules to be discovered were those involved in protein synthesis, mRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). In recent years, a vast number of additional RNA molecules have been identified. ‘RNA’ explains that these are non-coding RNAs that are not involved in protein synthesis, but influence many normal cellular and disease processes by regulating gene expression. RNA interference (RNAi) as one of the main ways in which gene expression is regulated is described with applications to therapy. Classes of RNA, including long non-coding RNAs and catalytic RNAs, are explained along with RNA techniques used to study RNA molecule and gene function.

Proteomic analysis of Dhh1 complexes reveals a role for Hsp40 chaperone Ydj1 in yeast P-body assembly

P-bodies (PB) are ribonucleoprotein (RNP) complexes that aggregate into cytoplasmic foci when cells are exposed to stress. While the conserved mRNA decay and translational repression machineries are known components of PB, how and why cells assemble RNP complexes into large foci remain unclear. Using mass spectrometry to analyze proteins immunoisolated with the core PB protein Dhh1, we show that a considerable number of proteins contain low-complexity (LC) sequences, similar to proteins highly represented in mammalian RNP granules. We also show that the Hsp40 chaperone Ydj1, which contains an LC domain and controls prion protein aggregation, is required for the formation of Dhh1-GFP foci upon glucose depletion. New classes of proteins that reproducibly co-enrich with Dhh1-GFP during PB induction include proteins involved in nucleotide or amino acid metabolism, glycolysis, tRNA aminoacylation, and protein folding. Many of these proteins have been shown to form foci in response to other stresses. Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins, suggesting an active role for RNA in PB function. Thus, global characterization of PB composition has uncovered proteins and RNA that are important for PB assembly.