Dicer-independent processing of short hairpin RNAs

Abstract

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

Figure 1.

Design of a set of shPol47 variants. The encoded siRNA sequence targeting the Pol region of HIV-1 is boxed. The name of each shRNA indicates the stem length and loop size, e.g. a stem length of 19 bp and a loop sequence of 5 nt was named 19/5.

Figure 2.

Knockdown activity of the 3′/5′ strands of the shPol47 variants. (A) The knockdown activity of the 3′ and the 5′ strands of the different shRNAs was determined by co-transfection of a luciferase reporter encoding either the sense- or antisense-target sequence, respectively. 293T cells were co-transfected with 25 ng of the respective firefly luciferase reporter plasmid, 0.5 ng of renilla luciferase plasmid and 5 ng of the corresponding shRNA constructs. An irrelevant shRNA (shNef) served as negative control, which was set at 100% luciferase expression. (B) Processing of the 3′ strand (upper panel) and 5′ strand (lower panel) of shPol47 was analysed by Northern blot analysis. HCT-116 cells were transfected with 5 μg of the shRNA constructs. An irrelevant shRNA (shGag) was used as negative control. Northern blot analysis was performed on total cellular RNA. The RNA size marker (nt) is shown on the left. The regular 21 nt siRNA products and the new ∼30 nt product are indicated (marked as *).

Figure 3.

Knockdown activity of the 3′/5′ strands of several shRT5 and shPol9 variants. (A) The knockdown activity of the 3′ and the 5′ strands of the indicated shRNAs was determined by co-transfection of a luciferase reporter encoding the sense (white bars) or antisense target sequence (black bars), respectively, in 293T cells. See Figure 2A for details. (B) Processing of the 3′/5′ strands of the indicated shRNAs was analysed by Northern blot analysis. We used LNA oligonucleotides to detect the 3′ (upper panel) and 5′ strand (lower panel) of the siRNA. An irrelevant shRNA (shGag) was used as negative control. The regular 21 nt products are marked and the * indicates the ∼30 nt RNA products.

Figure 4.

Design of additional shRT5 mutants varying in loop size and stem length. The shRT5 with a 19 bp stem and a 5-nt loop (19/5) was used as backbone for this design. The shRNA stem length was reduced/extended from the bottom of the hairpin, resulting in shRT5 variants 15/5–23/5. In addition, shRNA terminal loops ranging in size from 3 to 8 nt were designed (19/3–19/8) and the loop sequence was reversed (19/5R). The original shRT5 21/5A variant was also included.

Figure 5.

Knockdown activity of the shRT5 variants. RNAi activity of the 3′ side (lower panel) and 5′ side (upper panel) of the shRT5 was determined by co-transfection of a luciferase reporter encoding the sense or antisense target sequence, respectively, in 293T cells. See Figure 2A for details.

Figure 6.

The processing pattern and production of the 3′ and 5′ strand of the shRT5 variants. Processing of the 3′ strand (upper panel) and 5′ strand (lower panel) of shRT5 variants was analysed by Northern blot analysis. See Figure 2B for details.

Figure 7.

The short RNAs derived from the 3′ and 5′ strand of the shRT5 19/5 and 21/5A that associate with wild-type and mutant AGO2 proteins. AGO2-IP experiments were performed on cell lysates from HCT-116 cells co-transfected with shRT5 19/5 or 21/5A together with the wild-type (wt-AGO2), or mutant AGO2 proteins (N-AGO2 or cat-AGO2). The small RNAs associated with the AGO2 proteins were extracted and used for Northern blot analyses to detect the 3′ and 5′ strand of the shRNAs. The RNA size marker (nt) is shown on the left. The precursor shRNAs are indicated with a p and the new 33 or 37 nt product are indicated with *.

Figure 8.

Alternative shRNA processing mechanisms. Processing of shRNAs by the RNAi machinery can use two competing pathways, depending largely on the shRNA stem length. For shRNAs >19 bp, processing occurs via the conventional pathway in which the shRNA is cleaved by Dicer into an siRNA (19–21 nt) followed by RISC incorporation and AGO2-mediated cleavage of the target mRNA. In principle, each siRNA strand can instruct RISC for cleavage, but there is usually a strand preference (e.g. the 3′ side of the shRNA 21/5A). For shRNAs of 17–19 bp, processing occurs independent of Dicer. We refer to this design as AgoshRNA. Consistent with several literature reports, such minimal shRNA templates are not efficient substrates for Dicer (34,51). We propose that the AgoshRNA is loaded into RISC and cleaved by AGO2 on the 3′ side, resulting in the new 33 nt RNA product that, upon unfolding, can instruct target mRNA cleavage. RNAi is mediated by AGO2 cleavage of the antisense target mRNA. Thus, AGO2 is instrumental twice, for the shRNA processing (new function) and mRNA cleavage (old function). If the shRNA is <17 bp, no processing by the RNAi machinery occurs.