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Chemistry and Biology of Self-Cleaving Ribozymes.

ABSTRACT: Self-cleaving ribozymes were discovered 30 years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure, with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be used as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered.

SUBMITTER: Jimenez RM

PROVIDER: S-EPMC4630146 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Chemistry and Biology of Self-Cleaving Ribozymes.

Jimenez Randi M RM Polanco Julio A JA Lupták Andrej A

Trends in biochemical sciences 20151015 11

Self-cleaving ribozymes were discovered 30 years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure, with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be used as cofactors. Ribozymes have been discovered in hig ...[more]

PMID: 26481500

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Widespread occurrence of self-cleaving ribozymes.

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Scaling Catalytic Contributions of Small Self-Cleaving Ribozymes.

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Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes.

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SHAPE probing pictures Mg2+-dependent folding of small self-cleaving ribozymes.

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Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes.

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In vitro evolution of distinct self-cleaving ribozymes in diverse environments.

Project description:In vitro evolution experiments have long been used to evaluate the roles of RNA in both modern and ancient biology, and as a tool for biotechnology applications. The conditions under which these experiments have been conducted, however, do not reflect the range of cellular environments in modern biology or our understanding of chemical environments on the early earth, when the atmosphere and oceans were largely anoxic and soluble Fe(2+) was abundant. To test the impact of environmental factors relevant to RNA's potential role in the earliest forms of life, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the presence of either Fe(2+) or Mg(2+). Populations evolved under these different conditions are dominated by different sequences and secondary structures, demonstrating global differences in the underlying fitness landscapes. Comparisons between evolutionary outcomes and catalytic activities also indicate that Mg(2+) can readily take the place of Fe(2+) in supporting the catalysis of RNA cleavage at neutral pH, but not at lower pH. These results highlight the importance of considering the specific environments in which functional biopolymers evolve when evaluating their potential roles in the origin of life, extant biology, or biotechnology.

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High-throughput assay and engineering of self-cleaving ribozymes by sequencing.

Project description:Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes.

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Project description: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.

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Project description: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.

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