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Updated: Jun 15, 2026
Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis
Published on: March 30, 2019
Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced
Ian Sudbery1, Anton J Enright, Andrew G Fraser
1Work performed at: Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
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.
Insights
RNA interference (RNAi) screening using small interfering RNAs (siRNAs) can identify gene functions but is limited by off-target effects. This study found that specific seed sequences in siRNAs mimic microRNAs (miRNAs), causing unintended gene silencing and influencing TRAIL apoptosis pathways.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- RNA inhibition using small interfering RNAs (siRNAs) is a common method for gene function identification.
- Off-target effects, often due to siRNAs acting like microRNAs (miRNAs) via seed sequence matches, complicate RNAi screening results.
- This study investigated off-target effects in an siRNA screen for regulators of TRAIL apoptosis.
Purpose of the Study:
- To identify specific seed sequences responsible for off-target effects in siRNA screens.
- To determine the role of these seed sequences in regulating TRAIL apoptosis.
- To validate the presence of these seed sequences in known human miRNAs.
Main Methods:
- Conducted an siRNA screen to identify regulators of TRAIL apoptosis.
- Analyzed top-scoring siRNAs for over-represented hexamer and heptamer seed sequences.
- Utilized a statistical enrichment approach to systematically identify enriched seed sequences.
- Validated off-target effects by transfecting miRNA mimics.
Main Results:
- Identified 3 hexamer and 3 heptamer seed sequences enriched in top siRNAs.
- Systematically identified an additional 17 hexamer and 13 heptamer seed sequences.
- Demonstrated that the presence of specific seed sequences is sufficient to elicit a phenotype, explaining 6 of 8 confirmed off-target effects.
- Found that three enriched seed sequences are present in human miRNAs (miR-26a, miR-145, miR-384).
- Showed that mimics of these miRNAs protect cells from TRAIL-induced cell death.
Conclusions:
- Established a role for miR-26a, miR-145, and miR-384 in TRAIL-induced apoptosis.
- Confirmed that RNAi screening enriches for siRNAs with relevant off-target effects.
- Highlighted the utility of systematically analyzing enriched seed sequences to identify and understand siRNA off-target effects.
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01:22MicroRNAs
02:15Experimental RNAi
01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...