Visualizando a atividade nuclear do RNAi em células vivas humanas

quinta-feira, novembro 23, 2017

Visualizing nuclear RNAi activity in single living human cells

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

Author Affiliations

a The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;

b Institute of Nanotechnology, Bar-Ilan University, Ramat Gan 5290002, Israel;

c Department of Biochemistry & Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 6997801, Israel

Edited by Robert H. Singer, Albert Einstein College of Medicine, Bronx, NY, and approved September 11, 2017 (received for review May 4, 2017)


Significance

RNA interference (RNAi) is a natural process occurring in cells, and is used to silence genes. Typically, RNAi occurs via small RNA molecules generated in the cell nucleus, which are exported to the cytoplasm where they silence messenger RNA (mRNA) molecules. However, RNAi is thought to occur in the nucleus as well. To demonstrate that this process can occur in the nucleus and to determine its dynamics, we generated human cell systems that enabled us to image living cells and to track gene silencing as it transpired in real time. We found that the RNAi machinery can target the mRNA as it is being transcribed, and that silencing is mediated through modifications occurring on histone proteins bound to the DNA.

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.

transcription nuclear RNAi histone methylation live-cell imaging argonautes

Footnotes

1To whom correspondence should be addressed. Email: Yaron.Shav-Tal@biu.ac.il.

Author contributions: S.A., A.M., I.D., D.C., and Y.S.-T. designed research; S.A., A.M., I.D., S.T., and N.K. performed research; S.A., A.M., I.D., and I.K. analyzed data; and Y.S.-T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707440114/-/DCSupplemental.

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