Highly motif- and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression

Abstract

Recent bioinformatics studies have demonstrated a wide-spread occurrence of the hammerhead ribozyme (HHR) and similar small endonucleolytic RNA motifs in all domains of life. It is becoming increasingly evident that such ribozyme motifs participate in important genetic processes in diverse organisms. Although the HHR motif has been studied for more than three decades, only little is known about the consequences of ribozyme activity on gene expression. In the present study we analysed eight different naturally occurring HHR sequences in diverse genetic and organismal contexts. We investigated the influence of active ribozymes incorporated into mRNAs in mammalian, yeast and bacterial expression systems. The experiments show an unexpectedly high degree of organism-specific variability of ribozyme-mediated effects on gene expression. The presented findings demonstrate that ribozyme cleavage profoundly affect gene expression. However, the extent of this effect varies and depends strongly on the respective genetic context. The fast-cleaving type 3 HHRs [CChMVd(-) and sLTSV(-)] generally tended to cause the strongest effects on intracellular gene expression. The presented results are important in order to address potential functions of naturally occurring ribozymes in RNA processing and post-transcriptional regulation of gene expression. Additionally, our results are of interest for biotechnology and synthetic biology approaches that aim at the utilisation of self-cleaving ribozymes as widely applicable tools for controlling genetic processes.

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems

“…After normalizing the fold-reduction levels by accounting for the loss of gene expression, we observed that some ribozyme constructs (most notably the 5’UTR constructs) reduced gene expression more weakly compared to that data prior to normalization (Figures 2A/C). In general, the ribozymes/upstream competing sequences were observed to reduce gene expression more strongly in HEK293T cells compared to Drosophila embryos (Figures 2 and 3), which has also been observed in recent work (41). This difference could be due to different biological machinery between mammalian and insect models, different experimental assays, or the constructs themselves, as they contain different promoters and reporter genes.…”

“…mammalian vs. yeast) and experimental setups (e.g. in vitro vs. in vivo ) (41), which show that cellular context is likely important for the observed activity. Another possibility is that Mfold and Sfold are not accurately capturing RNA folding.…”

Simultaneous quantification of mRNA and protein in single cells reveals post-transcriptional effects of genetic variation

“…Therefore, the resulting signal is independent of the fate of the mRNA after gRNA release. Given the hammerhead ribozyme has a rate constant for self-cleavage of 1.5 per minute (Wurmthaler et al, 2018), gRNA abundance is not expected to reflect the half-life and stability of most yeast mRNAs, which have a median half-life of 3.6 minutes (Chan et al, 2018). By contrast, standard methods usually used in eQTL mapping quantify mRNA at steady state, which may explain some of the differences we observed between our mRNA-QTLs and known eQTLs identified by RNA sequencing.…”

Aptamer-based and aptazyme-based riboswitches in mammalian cells

“…However, it has been reported that ribozyme activity in living cells are not highly correlated among different cell types (bacteria, yeast, or mammalian cells) which is understandable considering the differences in translational mechanism, intracellular environment (RNA binding proteins, ribonucleases, etc. ), and mode of gene regulation by ribozymes in different cell types [17 ]. Because high-throughput screening of riboswitches directly in mammalian cells is technically challenging, alternative design strategies focusing on aptazymes that function in mammalian cells are desirable.…”