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Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing
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Visualizing nuclear RNAi activity in single living human cells.

Shira Avivi1,2, Amir Mor1,2, Iris Dotan3

  • 1The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.

Proceedings of the National Academy of Sciences of the United States of America
|October 27, 2017
PubMed
Summary

Nuclear RNA interference (RNAi) silences gene transcription in human cells. This process involves nascent messenger RNA (mRNA) and histone modifications, but not DNA methylation, confirmed via live-cell imaging.

Keywords:
argonauteshistone methylationlive-cell imagingnuclear RNAitranscription

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Gene Regulation

Background:

  • Nuclear RNA interference (RNAi) is a cellular mechanism that influences gene expression.
  • It can lead to transcriptional silencing, activation, or alternative splicing through chromatin modification.
  • The precise dynamics and mechanisms in living human cells remain incompletely understood.

Purpose of the Study:

  • To demonstrate and characterize nuclear RNAi-mediated transcriptional repression in real-time within single living human cells.
  • To elucidate the molecular pathways involved in nuclear RNAi-induced gene silencing.

Main Methods:

  • Utilized live-cell imaging techniques to observe nuclear RNAi dynamics.
  • Employed reporter gene systems with inducible promoters and short hairpin RNAs (shRNAs) targeting reporter mRNA.
  • Analyzed histone and DNA modifications associated with transcriptional changes.

Main Results:

  • Successfully detected and tracked nuclear RNAi-mediated transcriptional repression in living human cells.
  • Demonstrated that the silencing effect is mediated via nascent mRNA.
  • Identified the involvement of histone methylating enzymes in the silencing process.
  • Ruled out DNA methylation as a mechanism for this nuclear RNAi-induced silencing.

Conclusions:

  • Nuclear RNA interference is an active process in human cells that modulates transcription.
  • The mechanism involves nascent mRNA and histone modifications, highlighting a direct link between RNAi machinery and epigenetic regulation.
  • Live-cell imaging provides critical insights into the dynamic nature of nuclear RNAi.