Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution

Publication date: 18 April 2017
Source:Cell Reports, Volume 19, Issue 3
Author(s): Sethuramasundaram Pitchiaya, Laurie A. Heinicke, Jun I. Park, Elizabeth L. Cameron, Nils G. Walter
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-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 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.

Graphical abstract

Teaser

Pitchiaya et al. describe tools to interrogate gene-regulatory microRNAs inside living cells at single-molecule resolution. They find that the RNA silencing machinery and RNA targets mediate gene silencing surveillance by modulating the abundance and subcellular location of microRNAs. These findings and tools promise to facilitate single-cell screening of microRNA activity.


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