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Nucleic Acids Research Advance Access originally published online on September 18, 2009
Nucleic Acids Research 2009 37(20):6927-6941; doi:10.1093/nar/gkp735
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Nucleic Acids Research, 2009, Vol. 37, No. 20 6927-6941
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1

Timothy A. Vickers1,*, Walt F. Lima1, Hongjiang Wu1, Josh G. Nichols1, Peter S. Linsley2 and Stanley T. Crooke1

1Antisense Core Research, ISIS Pharmaceuticals and 2Regulus Therapeutics, Carlsbad, CA 92008, USA

*To whom correspondence should be addressed. Tel: +1 760 931 9200; Fax: +1 760 268 4989; Email: tvickers{at}isisph.com

Received March 9, 2009. Revised August 19, 2009. Accepted August 20, 2009.

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.


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