In Situ Fluorescence Analysis Demonstrates Active siRNA Exclusion From the Nucleus by Exportin 5

Nucleic Acids Res. 2006 Mar 6;34(5):1369-80. doi: 10.1093/nar/gkl001. Print 2006.

Abstract

Two types of short double-stranded RNA molecules, namely microRNAs (miRNAs) and short interfering RNAs (siRNAs), have emerged recently as important regulators of gene expression. Although these molecules show similar sizes and structural features, the mechanisms of action underlying their respective target silencing activities appear to differ: siRNAs act primarily through mRNA degradation, whereas most miRNAs appear to act primarily through translational inhibition. Our understanding of how these overlapping pathways are differentially regulated within the cell remains incomplete. In the present work, quantitative fluorescence microscopy was used to study how siRNAs are processed within human cells. We found that siRNAs are excluded from non-nucleolar areas of the nucleus in an Exportin-5 dependent process that specifically recognizes key structural features shared by these and other small RNAs such as miRNAs. We further established that the Exportin-5-based exclusion of siRNAs from the nucleus can, when Exp5 itself is inhibited, become a rate-limiting step for siRNA-induced silencing activity. Exportin 5 therefore represents a key point of intersection between the siRNA and miRNA pathways, and, as such, is of fundamental importance for the design and interpretation of RNA interference experimentation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Cell Nucleus / metabolism*
  • Fluorescent Dyes
  • HeLa Cells
  • Humans
  • Karyopherins / metabolism*
  • Microinjections
  • Microscopy, Fluorescence
  • RNA Interference*
  • RNA, Small Interfering / administration & dosage
  • RNA, Small Interfering / analysis
  • RNA, Small Interfering / metabolism*
  • Transfection

Substances

  • Fluorescent Dyes
  • Karyopherins
  • RNA, Small Interfering
  • XPO5 protein, human