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.
Project description:RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in C. elegans for the conserved RNA ligase RtcB, and show that RtcB ligates the xbp 1 mRNA during the IRE 1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. 4 paired-end RNA-seq reads. Control worms have pre-spliced tRNAs, RtcB-null have mutated RtcB, +/- tunicamycin treatment
Project description:RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in C. elegans for the conserved RNA ligase RtcB, and show that RtcB ligates the xbp 1 mRNA during the IRE 1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.
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).
Project description:To identify small RNA cleaved by RNaseL, we captured intracellular RNA with 2'-3' cyclic phosphates by ligation an Illumina-compatible adaptor and the RNA ligase RtcB.
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.
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.
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.
Project description:rs12-04_rnanonpolya - nad9 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 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. Control samples correspond to plants not induced with estradiol.
Project description:We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for stitch RNA).