Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1.
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.
Vickers
Timothy A
TA
Antisense Core Research, ISIS Pharmaceuticals, Carlsbad, CA 92008, USA. tvickers@isisph.com
Lima
Walt F
WF
Wu
Hongjiang
H
Nichols
Josh G
JG
Linsley
Peter S
PS
Crooke
Stanley T
ST
D000818:Animals; D060565:Argonaute Proteins; D002478:Cells, Cultured; D058534:Class I Phosphatidylinositol 3-Kinases; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism
D060565:Argonaute Proteins; D058534:Class I Phosphatidylinositol 3-Kinases; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D019869:Phosphatidylinositol 3-Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043244:Ribonuclease III; C115848:AGO1 protein, human; C526735:Ago2 protein, mouse; C497132:PIK3CB protein, human