Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution
Summary: 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 miR...
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doaj-952f1e7e38e64b1397ee482018315f372020-11-25T01:16:17ZengElsevierCell Reports2211-12472017-04-01193630642Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule ResolutionSethuramasundaram Pitchiaya0Laurie A. Heinicke1Jun I. Park2Elizabeth L. Cameron3Nils G. Walter4Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USASingle Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USASingle Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USASingle Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USASingle Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA; Corresponding authorSummary: 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. : 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. Keywords: microRNA, Argonaute, mRNA targets, anti-miRs, correlative counting analysis, single-molecule microscopyhttp://www.sciencedirect.com/science/article/pii/S2211124717304527 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Sethuramasundaram Pitchiaya Laurie A. Heinicke Jun I. Park Elizabeth L. Cameron Nils G. Walter |
spellingShingle |
Sethuramasundaram Pitchiaya Laurie A. Heinicke Jun I. Park Elizabeth L. Cameron Nils G. Walter Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution Cell Reports |
author_facet |
Sethuramasundaram Pitchiaya Laurie A. Heinicke Jun I. Park Elizabeth L. Cameron Nils G. Walter |
author_sort |
Sethuramasundaram Pitchiaya |
title |
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution |
title_short |
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution |
title_full |
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution |
title_fullStr |
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution |
title_full_unstemmed |
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution |
title_sort |
resolving subcellular mirna trafficking and turnover at single-molecule resolution |
publisher |
Elsevier |
series |
Cell Reports |
issn |
2211-1247 |
publishDate |
2017-04-01 |
description |
Summary: 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. : 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. Keywords: microRNA, Argonaute, mRNA targets, anti-miRs, correlative counting analysis, single-molecule microscopy |
url |
http://www.sciencedirect.com/science/article/pii/S2211124717304527 |
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