Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors

Abstract

Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3' position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi.

Figure 1.

Design of dual targeting lhRNAs. (A) Schematic representation of an lhRNA expression cassette showing upstream U6 promoter and the predicted structures of the transcribed lhRNAs. Dual targeting lhRNAs encode two 19 bp +2 nt siRNAs and have a 5 bp +2 nt spacer before the loop sequence. (B) Four different series of 48–50 bp lhRNAs were generated that target tat and nef as well as int and LTR. Each 19 bp +2 nt siRNA-encoding sequence is positioned either in the stem or loopside of the lhRNA duplex and separated by 1, 2 or 3 mismatched paired bases.

Figure 2.

Knockdown efficacy and processing of dual targeting lhRNAs. Dual luciferase reporter assays showing knockdown of the LTR and int targets (A) and tat and nef targets (B) when the target sequence was inserted downstream of the Renilla luciferase (hRLuc) open reading frame. Values represented are mean ratios of Renilla to Firefly luciferase (n = 3, SEM) and are normalized to cells transfected with a plasmid containing a U6 promoter only with no RNAi effector sequence (mock). Small RNA (PAGE) northern blot analysis was carried out on total RNA extracted from cells transfected with the indicated transcripts. Labelled probes complementary to the guide strand of LTR and int (C) or tat and nef (D) were hybridized to immobilized RNA and exposed to a phosphorimaging plate. lhRNA and shRNA precursor RNA as well as processed siRNAs are indicated. The amount of processed guide strand is shown and normalized for each blot relative to the shRNA (set at 100). Decade Marker^TM indicates fragment size and a probe complementary to small nuclear U6 RNA was used to detect U6 snRNA as a loading control.

Figure 3.

Generation of four effective individual siRNAs from a single dlhRNA transcript containing two adjacent lhRNAs. (A) Schematic representation of a dlhRNA expression cassette driven by a single promoter showing the predicted structure and derivation of four siRNAs. (B) Effective dual targeting long hairpin RNAs lhtat-nef +1 and lhLTR-int +1 were both combined in 5′ or 3′ positions within the dlhRNA transcript to generate lhLI-TN and lhTN-LI. (C) Low molecular weight northern blot analysis was carried out on total RNA extracted from cells transfected with the dlhRNA expression cassettes with individual targeting shRNAs used as positive controls. Labelled probes complementary to the guide and antisense strand of LTR, int, tat and nef were hybridized to immobilized RNA and exposed to a phosphorimaging plate. Precursor hairpin RNAs as well as processed siRNAs are indicated. The amount of processed guide strand is shown and normalized for each blot relative to the shRNA (set at 100). Decade Marker^TM indicates fragment size and a probe complementary to small nuclear U6 RNA was used to detect U6 snRNA as a loading control. (D) Dual luciferase reporter assays showing knockdown of the sense (S) and antisense (AS) targets of LTR, int, tat and nef when the target sequence was inserted downstream of the Renilla luciferase open reading frame. Values represented are mean ratios of Renilla to Firefly luciferase (n = 3, ± SEM) and are normalized to cells transfected with a plasmid containing a U6 promoter only with no RNAi effector sequence (mock).

Figure 4.

Inhibition of full-length HIV-1 molecular clone pNL4-3.Luc.R-E-. (A) The separate regions of the NL4-3.Luc sequence targeted by the four generated siRNAs are shown schematically. (B) HEK293 cells were transfected in a 1:1 ratio of pNL4-3.Luc.R-E- together with indicated hairpin constructs and trace amounts of Renilla luciferase plasmid phRL-CMV. The four siRNA-targeted regions are indicated above in the modified HIV genome of pNL4-3.Luc.R-E-. Values represented are mean ratios of Firefly luciferase normalized to Renilla luciferase (n = 3, ±SEM) and expressed as a percentage of mock (pU6 + 1) transfected cells (set at 100%).

Figure 5.

Assessment of non-specific effects mediated by long hairpin RNAs. (A) Analysis of effects of a shRNA, lhRNA and dlhRNA expression cassette on the repression of HBx target reporter sequence by an exogenously introduced miR-31 HBx shuttle using a dual luciferase assay. Co-transfection of reporter plasmid, containing an HBx target sequence downstream of the Renilla luciferase ORF, was carried out together with three different concentrations of RNAi expression cassettes and empty backbone plasmid (mock). Mean ratios of Renilla to Firefly luciferase (as a percentage of psiCheck2 empty backbone vector) were used to determine derepression of miR-31 HBx. Statistical significance was determined using a one-way ANOVA relative to mock transfected control (pU6 +1, *P < 0.05 and **P < 0.001). (B) The effect of hairpin expression cassettes on the function of endogenous miR-16 was analysed following co-transfection of a dual luciferase reporter plasmid containing seven copies of the miR-16 target downstream of the hRluc ORF together with the indicated hairpin-expressing plasmids or miR-16 sponge plasmid expressing seven copies of an imperfectly matched miR-16 target. Mean ratios of Renilla to Firefly luciferase were used to determine derepression of miR-16. (C) The induction of the IFN response was assessed by measuring IFN-β mRNA concentration in total RNA extracted from cells transfected with the indicated shRNA, lhRNA and multi-lhRNA expression cassettes. Poly I:C served as a positive control. Mean normalized ratios of IFN-β:GAPDH (n = 3, ± SEM) determined by using quantitative RT–PCR are indicated. Statistical significance was determined using a one-way ANOVA relative to mock transfected control (pU6 +1, *P < 0.001).