Engineered bacterial M1GS ribozyme efficiently cleaves the most abundant ribosomal RNA in a human cancer cell line

Ribonuclease P (RNase P), a ribozyme conserved across all domains of life, is involved in the tRNA 5′ maturation. RNase P recognizes precursor tRNA based on its structure, not the tRNA sequence. This feature had been exploited to engineer RNase P to selectively target and cleave any RNA as a gene inactivation strategy. Of these, a strategy called M1GS involves tethering an Escherichia coli M1 RNA to a short stretch of guide sequence (GS), which is complementary to the RNA targeted for cleavage. Despite its simplicity and versatility, M1GS tool appears to be underutilized compared to other gene inactivation strategies. Perhaps one of the reasons is that employment of the M1GS strategy requires prior knowledge about the requirements of the M1GS target sites. To facilitate its broader use, we have developed a Python script-based user-friendly bioinformatic tool built based on the requirements of M1GS to predict its target sites for any given RNA (using either DNA or RNA sequence as an input). In this study, we first demonstrate the utility of the bioinformatic tool in predicting M1GS target sites for human 28S rRNA and then we show that the customized M1GS-mediated downregulation of 28S rRNA in a human cancer cell line. We further validate the bioinformatic tool by predicting M1GS target sites for two previously targeted RNAs. Lastly, we discuss the utility of M1GS ribozyme-mediated rRNA downregulation as a potential anticancer modality in cancers where rRNAs are upregulated.