Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

  1. Nan Sook Lee1,2,
  2. Daniel H. Kim2,
  3. Jessica Alluin2,
  4. Marjorie Robbins2,
  5. Shuo Gu2,
  6. Haitang Li2,
  7. James Kim2,
  8. Paul M. Salvaterra3, and
  9. John J. Rossi2
  1. 1Division of Molecular Medicine, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA
  2. 2Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA
  3. 3Division of Neuroscience, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA

Abstract

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.

Keywords

Footnotes

  • Reprint requests to: John J. Rossi, Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA; e-mail: jrossi{at}coh.org; fax: (626)301-8271.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1014008.

    • Received January 24, 2008.
    • Accepted June 9, 2008.

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  1. Published in Advance August 12, 2008, doi: 10.1261/rna.1014008 RNA 14: 1823-1833 Copyright © 2008 RNA Society

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