siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.
Lin
Xiaoyu
X
Cancer Research, Global Pharmaceutical Research and Development, AP10, Abbott Laboratories 100 Abbott Park Road, Abbott Park, IL 60064, USA.
Ruan
Xiaoan
X
Anderson
Mark G
MG
McDowell
Jeffrey A
JA
Kroeger
Paul E
PE
Fesik
Stephen W
SW
Shen
Yu
Y
D000077329:Agammaglobulinaemia Tyrosine Kinase; D001483:Base Sequence; D002460:Cell Line; D015536:Down-Regulation; D054771:G-Protein-Coupled Receptor Kinase 4; D015723:Gene Library; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; D017346:Protein Serine-Threonine Kinases; D054771:G-Protein-Coupled Receptor Kinase 4; C517855:GRK4 protein, human