The MIME-seq2.0 technique utilizes a chimeric methyltransferase engineered to bind Argonaute proteins (hereafter referred to MIME enzyme) to protect the terminal sugar of small non-coding RNAs (sncRNAs) from oxidation, a reaction that inhibits unmodified RNA ligation, polyadenylation and cloning. In this study, we confirm that the MIME-seq2.0 technique efficiently distinguishes oxidized and non-oxidized sncRNAs. Expression of the MIME enzyme in the insulin-secreting cell line MIN6B1 protected miRNAs from oxidation and permitted their detection by RNA sequencing and quantitative PCR. Comparison of the sncRNA profile between wild type and MIME-expressing cells demonstrated protection from oxidation not only of miRNAs, but also of some Y-RNA and tRNA-derived fragments. Immunoprecipitation with Ago2 antibodies confirmed that the protected Y-RNA and tRNA fragments bind to Argonaute proteins. We also used this system to track miRNA transfer between cells via extracellular vesicles (EVs). miRNAs released inside EVs of MIME-expressing Jurkat T cells or C2C12 myotubes and delivered to MIN6B1 cells were protected from oxidation, enabling sensitive detection of the transferred miRNAs in the receiving cells. Overall, the MIME-seq2.0 technique provides a powerful tool for analyzing small RNA binding to Argonaute proteins in living cells and offers new possibilities for tracking RNA transfer across different cells.
Perrard et al. (Tue,) studied this question.