Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Numerous small hammerhead ribozyme variants associated with Penelope-like retrotransposons cleave RNA as dimers.

ABSTRACT: Hammerhead ribozymes represent the most common of the 9 natural classes of self-cleaving RNAs. The hammerhead catalytic core includes 11 highly-conserved nucleotides located largely within the unpaired regions of a junction formed by stems I, II and III. The vast majority of previously reported examples carry an additional pseudoknot or other tertiary interactions between nucleotides that precede stem I and nucleotides in the loop of stem II. These extra contacts are critical for high-speed RNA catalysis. Herein, we report the discovery of ?150,000 additional variant hammerhead representatives that exhibit diminished stem III substructures. These variants are frequently associated with Penelope-like retrotransposons, which are a type of mobile genetic element. Kinetic analyses indicate that these RNAs form dimers to cleave RNA.

SUBMITTER: Lunse CE

PROVIDER: S-EPMC5785218 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Numerous small hammerhead ribozyme variants associated with Penelope-like retrotransposons cleave RNA as dimers.

Lünse Christina E CE Weinberg Zasha Z Weinberg Zasha Z Breaker Ronald R RR

RNA biology 20171103 11

Hammerhead ribozymes represent the most common of the 9 natural classes of self-cleaving RNAs. The hammerhead catalytic core includes 11 highly-conserved nucleotides located largely within the unpaired regions of a junction formed by stems I, II and III. The vast majority of previously reported examples carry an additional pseudoknot or other tertiary interactions between nucleotides that precede stem I and nucleotides in the loop of stem II. These extra contacts are critical for high-speed RNA ...[more]

PMID: 27858507

Similar Datasets

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.

| S-EPMC4209133 | biostudies-literature

A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA.

Project description:Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5' UTR. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes are present in the 3' UTRs of rodent C-type lectin type II (Clec2) genes. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the polyadenylation signals within the 3' UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.

| S-EPMC2612532 | biostudies-literature

Development of a chimeric DNA-RNA hammerhead ribozyme targeting SARS virus.

Project description:OBJECTIVE:Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). METHOD:Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. RESULT:The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. CONCLUSION:These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS.

| S-EPMC7179559 | biostudies-literature

Solvent structure and hammerhead ribozyme catalysis.

Project description:Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

| S-EPMC2610685 | biostudies-literature

Cold denaturation of the hammerhead ribozyme.

Project description:Cold denaturation is a thermodynamic phenomenon resulting from a difference in the heat capacities, DeltaCp, of the folded and unfolded states of a macromolecule. Whereas this phenomenon has been extensively studied in proteins, it has been thought not to occur in nucleic acids due to a negligible DeltaCp of folding. Questioning the validity of this assumption, the low-temperature structure of the hammerhead ribozyme, a small catalytic RNA, was investigated by circular dichroism spectroscopy. In the presence of 10 mM Mg2+ at pH 5.0 and 40% methanol, a cold unfolding event likely corresponding to tertiary structure loss was observed with a Tm of -20 degrees C. In 500 mM NaCl at pH 6.6, and 40% methanol, large-scale unfolding of the ribozyme at both hot (Tm = 53 degrees C) and cold (Tm = -1 degrees C) temperatures occurred. Fitting of these data to a two-state model allowed determination of DeltaCp = 3.4 kJ mol-1 K-1, corresponding to >/=0.18 kJ K-1 (mol base pair)-1, in good agreement with recently published calorimetric values for DNA duplexes. These results constitute the first direct observation of cold denaturation of a nucleic acid, and point to the importance of DeltaCp terms in the thermodynamics of nucleic acid folding.

| S-EPMC3071246 | biostudies-literature

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.

| S-EPMC2948978 | biostudies-literature

Efficient ligation of the Schistosoma hammerhead ribozyme.

Project description:The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

| S-EPMC3203546 | biostudies-literature

Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR.

Project description:Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. We report that the HDV ribozyme sequence alone can activate PKR. To elucidate the RNA structural basis for this, we prepared a number of HDV variants, including those with shortened or lengthened P4 pairing regions, with the anticipation that lengthening the P4 extension would yield a more potent activator since it would offer more base pairs of dsRNA. Surprisingly, the variant with a shortened P4 was the most potent activator. Through native gel mobility and enzymatic structure mapping experiments we implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease.

| S-EPMC3504668 | biostudies-literature

Small Molecule Recognition Triggers Secondary and Tertiary Interactions in DNA Folding and Hammerhead Ribozyme Catalysis.

Project description:We have identified tris(2-aminoethyl)amine (tren)-derived scaffolds with two (t2M) or four (t4M) melamine rings that can target oligo T/U domains in DNA/RNA. Unstructured T-rich DNAs cooperatively fold with the tren derivatives to form hairpin-like structures. Both t2M and t4M act as functional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a U-rich sequence. Catalysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refolding of stem secondary structure or tertiary bridging interactions between loop and stem. The simplicity of the t2M/t4M binding site enables programming of allostery in RNAs, recoding oligo-U domains as potential sites for secondary structure or tertiary contact. In combination with a facile and general method for installation of the t2M motif on primary amines, the method described herein streamlines design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.

| S-EPMC5938730 | biostudies-literature

The hammerhead ribozyme: structure, catalysis, and gene regulation.

Project description:The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme's active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2'-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3'-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.

| S-EPMC4008931 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data