Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs

Abstract

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity

“…We suggest that the design of shRNA-based studies should involve careful consideration of several parameters, such as shRNA dose (Grimm, 2011), promoter choice (Lebbink et al, 2011;Sun et al, 2013), AAV serotype (Ehlert et al, 2010) and construct backbone (Boudreau et al, 2009;Han et al, 2011;McBride et al, 2008). These factors should subsequently be tested with respect to titer, construct and specificity in vivo.…”

Guidelines for the optimal design of miRNA-based shRNAs

“…On the contrary, the U6 promoter is known to be robust and possess a more precise start. Thus, U6-driven shRNAs are more potent, compared to those transcribed by Pol II, in a manner that is dependent on cellular context [24]. …”

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

“…EBV miRNA clusters were expressed from the intron of the pLenti-Blast-eGFPintron vector [40]. The Tough Decoy expression vector pSicoR-EF1a-ZeoR-T2A-mAmetrine (Figure 1(a)) was derived from pSicoR-EF1a-PuroR-T2A-mCherry [44] by replacing the PuroR-T2A-mCherry cassette for a ZeoR-T2A-mAmetrine cassette. TuDs were cloned immediately downstream of the mouse U6 promoter (sequences listed in Table S1).…”