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The MIME-seq technique allows to monitor the interaction of small non-coding RNAs with Argonaute proteins and their transfer to other cells

ABSTRACT: The MIME-seq2.0 technique, developed by Mandlbauer et al., utilizes a chimeric HEN1 methyltransferase engineered to bind to Argonaute proteins (MIME enzyme) to protect the terminal sugar of small non-coding RNAs (sncRNAs) from oxidation, a reaction that inhibits RNA ligation and cloning. In this study, we confirm that the MIME-seq technique efficiently distinguish oxidized and non-oxidized sncRNAs using RNA sequencing and quantitative PCR. Upon transfection of the insulin-secreting cell line MIN6B1 with or without the MIME enzyme, we observed a significant loss in the detection of miRNAs following RNA oxidation, with a strong recovery rate in MIME-expressing cells. Comparison of the sncRNA profile in 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 in EVs of MIME-expressing Jurkat T cells or C2C12 myotubes and delivered to MIN6B1 cells were protected from oxidation, enabling sensitive and specific detection of the transferred miRNAs in the receiving cells. Overall, the MIME-seq technique provides a powerful tool for analyzing RNA methylation and Argonaute binding in living cells and offers new possibilities for tracking RNA transfer across different cell types.

ORGANISM(S): Mus musculus

PROVIDER: GSE313744 | GEO | 2026/05/12

REPOSITORIES: GEO

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