Abstract
Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in ninemammalian transcripts, we show that our assayreliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides insights into sequence requirements of effective RNAi.
Highlights
RNA interference (RNAi) provides a programmable mechanism for targeted suppression of gene expression
Single-Vector Sensor Assay for Functional short hairpin RNAs (shRNAs) Evaluation Synthetic RNAi triggers can be accurately evaluated in functional assays by placing their cognate target site (‘‘Sensor’’) in the 30UTR of a reporter gene and quantifying its RNAi-mediated repression (Du et al, 2004; Kumar et al, 2003)
We reasoned that physically linking shRNAs and their cognate target sites in a single vector would enable multiplexed analysis of shRNA-target pairs
Summary
RNA interference (RNAi) provides a programmable mechanism for targeted suppression of gene expression. Through a highly conserved pathway, the RNAi machinery recognizes and processes double-stranded RNAs into small RNAs that guide the repression of complementary genes (for review, see Bartel, 2004; Hannon, 2002). Most miRNAs are produced through a coordinated processing program whereby primary miRNA transcripts (pri-miRNAs) are cleaved by the nuclear Drosha/DGCR8 complex, resulting in the formation of precursor miRNAs (pre-miRNAs). These short hairpin-like molecules are actively exported to the cytoplasm, where Dicer excises mature small RNA duplexes that are incorporated into the RNA-induced silencing complex (RISC). AGO2 discards the passenger (Leuschner et al, 2006; Matranga et al, 2005) and uses the guide for selection of complementary target mRNA substrates, whose expression is suppressed by accelerated mRNA degradation and/or translational inhibition
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
