Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution

作者：Pitchiaya Sethuramasundaram; Heinicke Laurie A; Park Jun I; Cameron Elizabeth L; Walter Nils G^*

来源：Cell Reports, 2017, 19(3): 630-642.

DOI：10.1016/j.celrep.2017.03.075

Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence- based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecularweight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereasmiRNAstrand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs.