Visualizing nuclear RNAi activity in single living human cells

Shira Avivi, Amir Mor, Iris Dotan, Sivan Tzadok, Itamar Kanter, Noa Kinor, Dan Canaani, Yaron Shav-Tal

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Nuclear RNA interference (RNAi) is mediated by the canonical RNAi machinery and can lead to transcriptional silencing, transcriptional activation, or modulation of alternative splicing patterns. These effects transpire through changes in histone and DNA modifications via RNAi-mediated recruitment of chromatin-modifying enzymes. To prove that nuclear RNAi occurs and modulates transcription in human cells, we used live-cell imaging to detect and track nuclear RNAi transcriptional repression in single living human cells. While employing reporter genes constructed with inducible promoters and cognate-inducible short hairpin RNA (shRNA) targeted against the reporter coding region, we have characterized the dynamics of the nuclear RNAi process in living human cells. We show that the silencing effect is mediated through the nascent mRNA, followed by activity of histone methylating enzymes, but not through DNA methylation.

Original languageEnglish
Pages (from-to)E8837-E8846
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number42
DOIs
StatePublished - 17 Oct 2017

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. We thank Oded Singer (Weizmann Institute) and Didier Trono (EPFL) for reagents. We are grateful to Shulamit Michaeli, Vaibhav Chikne, and K. Shanmugha Rajan [Bar-Ilan University (BIU)] for assistance and advice on quantifying siRNA levels, and to Avi Jacob (BIU) for helping with image analysis. Y.S.-T. was supported by the Israel Science Foundation and the European Research Council. D.C. was supported by the Orgler Fund for Cancer Genetics Research.

Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.

Keywords

  • Argonautes
  • Histone methylation
  • Live-cell imaging
  • Nuclear RNAi
  • Transcription

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