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<p>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.</p>

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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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月17日

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  • Jérôme Perrard,
  • Claudiane Guay,
  • Nadège Zanou,
  • Romano Regazzi

摘要

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.