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Single-molecule imaging of microRNA-mediated gene silencing in cells

Author

Listed:
  • Hotaka Kobayashi

    (Albert Einstein College of Medicine
    PRESTO, Japan Science and Technology Agency
    The University of Tokyo)

  • Robert H. Singer

    (Albert Einstein College of Medicine)

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying miRNA function at the single-molecule level with spatiotemporal information inside cells.

Suggested Citation

  • Hotaka Kobayashi & Robert H. Singer, 2022. "Single-molecule imaging of microRNA-mediated gene silencing in cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-29046-5
    DOI: 10.1038/s41467-022-29046-5
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    References listed on IDEAS

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    1. Rajagopal, 2014. "The Human Factors," Palgrave Macmillan Books, in: Architecting Enterprise, chapter 9, pages 225-249, Palgrave Macmillan.
    2. Lee P. Lim & Nelson C. Lau & Philip Garrett-Engele & Andrew Grimson & Janell M. Schelter & John Castle & David P. Bartel & Peter S. Linsley & Jason M. Johnson, 2005. "Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs," Nature, Nature, vol. 433(7027), pages 769-773, February.
    3. Nayi Wang & Ji Zheng & Zhuo Chen & Yang Liu & Burak Dura & Minsuk Kwak & Juliana Xavier-Ferrucio & Yi-Chien Lu & Miaomiao Zhang & Christine Roden & Jijun Cheng & Diane S. Krause & Ye Ding & Rong Fan &, 2019. "Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
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    Cited by:

    1. Charlotte A. Cialek & Gabriel Galindo & Tatsuya Morisaki & Ning Zhao & Taiowa A. Montgomery & Timothy J. Stasevich, 2022. "Imaging translational control by Argonaute with single-molecule resolution in live cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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