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

Abstract

M1GS, an engineered Ribonuclease P (RNase P), can be customized to specifically target and cleave any RNA. It involves tethering an Escherichia coli M1 RNA to a short guide sequence (GS) complementary to the RNA targeted for cleavage. We have developed a Python script-based bioinformatic tool built based on the requirements of M1GS, coupled to secondary structure predictions, to identify M1GS target sites for any given RNA. Primarily, we demonstrate the utility of the tool for predicting M1GS target sites across multiple RNAs, including human 28S ribosomal RNA (rRNA), and then we demonstrate efficient M1GS-mediated downregulation of 28S rRNA in a human cancer cell line. Efficient cleavage of rRNA, the most abundant RNA molecule, suggests a high turnover number for the M1GS ribozyme. Lastly, we discuss the utility of M1GS ribozyme-mediated rRNA downregulation as a potential anticancer modality in cancers where rRNAs are upregulated.