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Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium.

ABSTRACT: Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribozymes is highly conserved, which could contribute to a preference of hammerhead ribozymes toward certain cations. Here, we show a naturally occurring variation in the catalytic core of hammerhead ribozymes, A6C, that can favor one metallic ion, Mn2+, over several other cations.

SUBMITTER: Naghdi MR

PROVIDER: S-EPMC7151607 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium.

Naghdi Mohammad Reza MR Boutet Emilie E Mucha Clarisse C Ouellet Jonathan J Perreault Jonathan J

Non-coding RNA 20200320 1

Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribo ...[more]

PMID: 32245091

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Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum.

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Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

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Project description:By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg(2+)) ions, proceeds via an in-line-attack by CYT 17 O2' on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack.

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Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus.

Project description:The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

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Computational mutagenesis studies of hammerhead ribozyme catalysis.

Project description:Computational studies of the mutational effects at the C3, G8, and G5 positions of the hammerhead ribozyme (HHR) are reported, based on a series of twenty-four 100-ns molecular dynamics simulations of the native and mutated HHR in the reactant state and in an activated precursor state (G8:2'OH deprotonated). Invoking the assumptions that G12 acts as the general base while the 2'OH of G8 acts as a general acid, the simulations are able to explain the origins of experimentally observed mutational effects, including several that are not easily inferred from the crystal structure. Simulations suggest that the Watson-Crick base-pairing between G8 and C3, the hydrogen bond network between C17 and G5, and the base stacking interactions between G8 and C1.1, collectively, are key to maintaining an active site structure conducive for catalytic activity. Mutation-induced disruption of any of these interactions will adversely affect activity. The simulation results predict that the C3U/G8D double mutant, where D is 2,6-diaminopurine, will have a rescue effect relative to the corresponding single mutations. Two general conclusions about the simulations emerge from this work. First, mutation simulations may require 30 ns or more to suitably relax such that the mutational effects become apparent. Second, in some cases, it is necessary to look beyond the reactant state in order to interpret mutational effects in terms of catalytically active structure. The present simulation results lead to better understanding of the origin of experimental mutational effects and provide insight into the key conserved features necessary to maintain the integrity of the active site architecture.

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Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs.

Project description:Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes.

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