Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1105081 | ENA |
REPOSITORIES: ENA
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Project description:Like other functional RNAs, ribozymes contain a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-peripheral interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (ClvSeq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. Most deleterious mutations impaired RNA self-assembly. All-atom MD simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop rewire the network of hydrogen bonds around the catalytic site. Remarkably, IL4 mutations introduce a non-native helix interface that corrupts folding of the wild type core, eliminating activity. The results illustrate how competition between native and non-native structures in RNA drives the natural selection of central and peripheral tertiary interaction motifs.
2024-09-30 | GSE261357 | GEO
Project description:Naturally-occurring catalytic RNA molecules — ribozymes — have attracted a great deal of research interest, yet very few of them have been identified in humans. Here, we developed a genome-wide approach to discover self-cleaving ribozymes and identified one naturally-occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to yet un-identified class of small self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path from appearance between ~130 and ~65 million years ago (mya) to gain of self-cleavage activity very recently, ~13–10 mya, in the common ancestor of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within an lncRNA belonging to the class of very long intergenic non-coding (vlinc) RNAs. The presence of a catalytic RNA enzyme in lncRNA opens a possibility that these transcripts could function by carrying catalytic RNA domains.
2020-12-19 | GSE163477 | GEO
Project description:rs12-06_a9-mrnonpolya - matr knockdown 1 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the matR mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the matR mRNA knockdown on the overall plant transcriptome. Control plants express either no ribozyme (C0a and C0b) or the shuttle RNA combined with a ribozyme that has no specific target in A. thaliana (SD).
2019-03-01 | GSE127499 | GEO
Project description:As part of an examination of a newly-evolved RNA polymerase ribozyme, 38-6, products of primer extension experiments using an RNA template encoding the hammerhead RNA endonuclease ribozyme, using 38-6 and its less active ancestor 24-3. These products were analyzed by next-generation sequencing to determine the rates of substitution, deletion, and insertion mutations for both polymerases.
2019-12-17 | GSE142114 | GEO
Project description:Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3M-bM-^@M-2 untranslated region (3M-bM-^@M-2-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery. mRNA profiles were generated from 3D7 wild-type and DHFR-TS-GFP_glmS integrant parasites in untreated and treated with 10 mM Glucosamine conditions in duplicate.
2013-09-03 | E-GEOD-43125 | biostudies-arrayexpress
Project description:rs13-02_mitomanip - mitomanip microarray experiment 4 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the nad9 mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the nad9 mRNA knockdown on the overall plant transcriptome is analyzed over 4 successive days. Control plants express the shuttle RNA combined with a ribozyme that has no specific target in A. thaliana.
2019-03-04 | GSE127756 | GEO
Project description:rs14-01_mitomanip2 - microarray experiment 4 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the nad9 mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the nad9 mRNA knockdown on the overall plant transcriptome is analyzed over 4 successive days. Control plants express the shuttle RNA combined with a ribozyme that has no specific target in A. thaliana.
2017-01-05 | GSE93122 | GEO
Project description:The sequence determinants of chromatin bivalency remain unclear. We analysed sequence determinants of chromatin bivalency genome-wide in several mammalian species and performed a series of transgenic experiments in mouse ES cells. Genome-wide mapping of H3K27me3 in rat ES cells and ChIP-seq with anti-Ezh2 antibody in transgenic mouse ES cells
2011-11-16 | E-GEOD-27580 | biostudies-arrayexpress
Project description:We solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify structural changes that evolutionarily rendered hAGO1 inactive, we compared our structure with published structures of guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop and thereby forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully-paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad (R805H) led to low level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions (P670S and P675Q) substantially augmented hAGO1 activity. Evolutionary changes to hAGO1 were subtle and readily reversible, suggesting that loading of guide RNA and seed-based miRNA-target RNA pairing constrain its sequence drift.
2013-06-20 | GSE48076 | GEO
Project description:The sequence determinants of chromatin bivalency remain unclear. We analysed sequence determinants of chromatin bivalency genome-wide in several mammalian species and performed a series of transgenic experiments in mouse ES cells.
2011-11-16 | GSE27580 | GEO