SUMMARY Regulation of 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 sub-cellular 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 molecular weight complexes and rendered repression-incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand 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 sub-cellular 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.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for “the development of super-resolved fluorescence microscopy” based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a “Principles of Single Molecule Techniques 2014” course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.