Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis.
BACKGROUND
RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s).


RESULTS
We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death.


CONCLUSIONS
We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.
Sudbery
Ian
I
Work performed at: Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
Enright
Anton J
AJ
Fraser
Andrew G
AG
Dunham
Ian
I
D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D034741:RNA, Small Interfering / Q000032:analysis* / Q000235:genetics; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*
D035683:MicroRNAs; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C541010:MIRN145 microRNA, human; C528163:MIRN26 microRNA, human