Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis

BMC Genomics. 2010 Mar 15:11:175. doi: 10.1186/1471-2164-11-175.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis*
  • Base Sequence
  • Cell Line, Tumor
  • Humans
  • MicroRNAs / genetics*
  • RNA, Small Interfering / analysis*
  • RNA, Small Interfering / genetics
  • Signal Transduction
  • TNF-Related Apoptosis-Inducing Ligand / metabolism*

Substances

  • MIRN145 microRNA, human
  • MIRN26A microRNA, human
  • MicroRNAs
  • RNA, Small Interfering
  • TNF-Related Apoptosis-Inducing Ligand