Evaluating the susceptibility of AGO2-loaded microRNAs to degradation by nucleases in vitro

Publication date: Available online 17 May 2018 Source:Methods Author(s): Reyad Elbarbary, Lynne E. Maquat MicroRNAs (miRNAs) comprise a class of small non-coding RNAs that regulate the stability and/or translatability of most protein-coding transcripts. Steady-state levels of mature miRNAs can be controlled through mechanisms that influence their biogenesis and/or decay rates. Pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway that promotes the sequence-specific endonucleolytic decay of miRNAs that harbor a CA and/or UA dinucleotide. Here, we describe an in vitro assay for evaluating the susceptibility of AGO2-loaded miRNAs to degradation by different classes of nucleases. This in vitro approach can be used to complement in vivo studies that aim to identify novel miRNA decay factors.
Source: Methods - Category: Molecular Biology Source Type: research