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. 2015;12(1):92-100.
doi: 10.1080/15476286.2015.1017204.

Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs

Ying Poi Liu  1 , Margarete Karg, Alex Harwig, Elena Herrera-Carrillo, Aldo Jongejan, Antoine van Kampen, Ben Berkhout
Affiliations

Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs

Ying Poi Liu et al. RNA Biol. 2015.

Abstract

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.

Keywords: AgoshRNA; Argonaute 2; Dicer; RNA processing; shRNA.

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Figures

Figure 1.
Figure 1.
Canonical (Dicer-mediated) and non-canonical (AGO-2 mediated) paths for shRNA and miRNA processing (A) Canonical Dicer-mediated processing of shRNAs and pre-miRNAs. Top: a regular short hairpin RNA (shRNA) of 21 bp in length is processed by Dicer into siRNAs. Bottom: a regular pre-miRNA is processed by Dicer into the mature miRNA duplex. The guide strand (thick line) of the siRNA/miRNA duplex will subsequently be incorporated into RISC to induce RNAi silencing. (B) Non-canonical AGO2-mediated processing of AgoshRNAs and miR-451. Top: AgoshRNAs are hairpins of only 19 bp with a small terminal loop that bypass Dicer-processing and undergo cleavage by AGO2 between bp 10 and 11 on the 3′ side of the duplex (◂). Bottom: miR-451 is the founding member of a class of Dicer-independent, AGO2-processed miRNAs. A thick line represents the guide strand. Hairpin characteristics shared with the AgoshRNA design are the short stem and small loop. The miR-451 is processed further by tailing and trimming as indicated.
Figure 2.
Figure 2.
Design of AgoshRNA cleavage site mutants The wild-type (wt) AgoshRNA is based on the AgoshRT5 hairpin. The AGO2 cleavage site is marked by an arrow. Mutants 1–5 have one or multiple mismatches introduced near the cleavage site. The mutated nt are marked in a gray box. The thermodynamic stability of the wt and mutant hairpins is indicated below (ΔG in kcal/mole). Deep sequencing of RNA mutant 1 products revealed an additional 5′-terminal C residue due to a-1 shift of the transcriptional start site in the H1 promoter.
Figure 3.
Figure 3.
Northern blot analysis of the AgoshRNA cleavage site mutants An irrelevant shRNA (shNef) and Bluescript (pBS) were used as negative controls. The regular 21 nt shRNA products are indicated and the special AGO2-dependent ∼30 nt AgoshRNA products are marked (*). Lane M contains an RNA size ladder. The hairpin side that was probed is marked in the cartoon on the right.
Figure 4.
Figure 4.
Transcription start site usage in AgoshRNA constructs. SOLiD deep sequencing was performed on the wt and mut 1, 2 or 3 AgoshRT5 constructs. The natural starting position is marked as +1.
Figure 5.
Figure 5.
Knockdown activity of the AgoshRNA cleavage site mutants (A) The knockdown activity of the indicated AgoshRNAs was determined by co-transfection of 25 ng of hairpin construct with 100 ng of a luciferase reporter. This reporter encoded either the sense or antisense target sequence, which monitors the activity of the AgoshRNA 3′ or 5′ strand, respectively. Luciferase values measured for the pBS control were set at 100%. The mean values and standard deviations are based on 3 independent experiments. (B) The AgoshRNA knockdown activity (5′ strand) was carefully determined by transfecting different amounts of the indicated constructs (1.25, 5 and 20 ng) together with a fixed amount (100 ng) of the antisense reporter. For each condition, luciferase values obtained for pBS were set at 100%. The averages and standard deviations of 3 independent transfections are shown.
Figure 6.
Figure 6.
Probing the ends of a regular shRNA versus AgoshRNA molecule. SOLiD deep sequencing was performed on small RNAs isolated from shRT5 (21/5) or AgoshRT5 (19/5) expressing cells. (A) The predicted structure of the short hairpin shRT5. The predicted 2-nt staggered Dicer cleavage site is indicated with a line. Depicted are the percentages of the total reads that either end (5′ side) or start (3′ side) around the Dicer cleavage-site. The cut off was set at 5%. These percentages are also presented in a bar-graph. (B) Depicted on the predicted structure of AgoshRT5 are the percentages of the total reads that end around the AGO2 cleavage site on the 3′ side of the hairpin. The cut off was set at 1%. These percentages are also depicted in a bar-graph. The results for mutants 1 and 3 are shown as inserts.
Figure 7.
Figure 7.
Processing of the 3′/5′ strands of AgoshRNAs in the presence of a complementary target RNA. HEK 293T cells were transfected with regular shRNA (21/5 and 22/5) or AgoshRNA (19/3 and 19/5) constructs and a reporter construct encoding a transcript with a complementary target sequence. As a control, a reporter encoding an irrelevant target sequence (shNef) was used. Total RNA was isolated from the transfected cells and subjected to RNA gel blot analysis to analyze the cleavage products and putative modification by tailing and/or trimming. Lane M contains an RNA size ladder. The hairpin side that was probed is marked in the cartoon on the right.
Figure 8.
Figure 8.
AgoshRNA activity in Dicer-deficient cells HCT-116 cells that express the wt Dicer protein and a non-functional Dicer protein (Dcr-) were transfected with the regularly Dicer-processed shRNA construct (21/5) or the AGO2-processed AgoshRNA construct (19/5) together with a luciferase reporter encoding the sense or antisense target sequence, respectively. Activity scored in transfection of the luciferase reporter with the control plasmid pBS was set at 100%. The averages and standard deviations of 3 independent transfections are shown.
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