Hepatitis delta virus (HDV) and cytoplasmic polyadenylation element-binding protein 3 (CPEB3) ribozymes form a family of self-cleaving RNAs characterized by a conserved nested double-pseudoknot and minimal sequence conservation. Secondary structure-based searches were used to identify sequences capable of forming this fold, and their self-cleavage activity was confirmed in vitro. Active sequences were uncovered in several marine organisms, two nematodes, an arthropod, a bacterium, and an insect virus, often in multiple sequence families and copies. Sequence searches based on identified ribozymes showed that plants, fungi, and a unicellular eukaryote also harbor the ribozymes. In Anopheles gambiae, the ribozymes were found differentially expressed and self-cleaved at basic developmental stages. Our results indicate that HDV-like ribozymes are abundant in nature and suggest that self-cleaving RNAs may play a variety of biological roles.

Background: HDV-like ribozymes map to several non-LTR retrotransposons, although their roles are not fully understood. Results: Self-cleaving ribozymes are found widespread in retrotransposons and promote translation initiation in vitro and in vivo. Conclusion: Ribozymes process many non-LTRs and facilitate translation of their ORFs. Significance: These new roles further explain the retrotransposon cycle and expand the functions of catalytic RNA.

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

Many non‐long terminal repeat (non‐LTR) retrotransposons lack internal promoters and are co‐transcribed with their host genes. These transcripts need to be liberated before inserting into new loci. Using structure‐based bioinformatics, we have recently shown that several classes of retrotransposons in phyla spanning arthropods, nematodes, and chordates utilize self‐cleaving ribozymes of the hepatitis delta virus (HDV) family for processing their 5′ termini. Ribozyme‐terminated retrotransposons include rDNA‐specific R2, R4 and R6; telomere‐specific SART; and Baggins and RTE elements. The R2 and R6 ribozymes were recently shown to promote translation initiation of downstream open reading frames in translation reactions in vitro with rabbit reticulocyte lysate and they also demonstrate in vivo activity in Drosophila S2 cell transfections. Translation initiation now extends to other retrotransposon‐associated ribozymes including those that do not insert site‐specifically. Additionally, mutagenesis and deletion experiments have been used to establish the functions of various parts of the ribozyme in ribosome binding and translation. Novel roles, such as those presented here, highlight the biological importance of self‐cleaving ribozymes as well as functional RNAs in general.

Using structure‐based bioinformatics, we recently discovered self‐cleaving ribozymes of the Hepatitis Delta Virus (HDV) family in another virus, a bacterium, and many eukaryotes. The genomic locations of some of these ribozymes suggest that they play a variety of biological roles. HDV‐like ribozymes from several organisms are associated with putative reverse transcriptase genes and retrotransposons, pointing to a possible function in retrotransposition. The specific roles of HDV‐like ribozymes in retrotransposition are being investigated using a variety of in vitro and in vivo approaches.