The Loop Position of shRNAs and Pre-miRNAs Is Critical for the Accuracy of Dicer Processing In Vivo

• Published in: Cell 2012-11, Vol.151 (4), p.900-911
• Main Authors: Gu, Shuo, Jin, Lan, Zhang, Yue, Huang, Yong, Zhang, Feijie, Valdmanis, Paul N., Kay, Mark A.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Embryo, Mammalian - cytology; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; mammals; Mice; microRNA; MicroRNAs; Ribonuclease III - metabolism; RNA interference; RNA, Small Interfering - chemistry; RNA, Small Interfering - metabolism; Sequence Analysis, RNA; small interfering RNA; therapeutics
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2012.09.042

Short hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that, in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5′/3′ counting rules. These promiscuous noncanonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ∼1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a “loop-counting rule,” we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies. [Display omitted] ► Cleavage of shRNAs by Dicer is heterogeneous, causing potential off-target effects ► Noncanonical Dicer cleavage products are independent of the substrate RNA ends ► Dicer uses a loop-counting rule to process shRNAs/pre-miRNAs 2 nt from a bulge ► This rule can be used to design therapeutic RNAs with reduced off-target effects A loop or bulge two nucleotides upstream of the mature siRNA or microRNA directs precise processing of the hairpin precursor by Dicer. Using this “loop-counting rule,” therapeutic hairpin RNAs that induce robust target silencing with reduced off-target effects can be designed.