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. 2017 Aug 15;12(8):e0183269.
doi: 10.1371/journal.pone.0183269. eCollection 2017.

Analysis of AgoshRNA maturation and loading into Ago2

Alex Harwig  1 , Zita Kruize  1 , Zhenhuang Yang  2 , Tobias Restle  2 , Ben Berkhout  1
Affiliations

Analysis of AgoshRNA maturation and loading into Ago2

Alex Harwig et al. PLoS One. .

Abstract

The RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that-after Ago2 cleavage-AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA in vitro. In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Processing and Ago2 loading of shRNA and AgoshRNA molecules.
Illustration of the processing options for the Dicer-dependent shRNA and Dicer-independent AgoshRNA. Short hairpin RNAs are cleaved by Dicer resulting in a ds siRNA molecule. One of the strands is selected to be loaded into Ago2. It is however unknown whether the siRNA enters Ago as duplex, after which the passenger strand is degraded (duplex loading model), or whether one of the strands is selected prior to Ago loading by an unknown helicase (helicase model). On the other hand, the Dicer-independent AgoshRNA (and miR-451) are too small to be cleaved by Dicer and are loaded in Ago2 in their precursor form (pre-Agosh). The 5’ end can dock into the Ago2 MID-domain (closed circle), but the 3’ end is not in the vicinity of the Ago2 PAZ-domain and thus unable to dock (open circle). The hairpin is subsequently cleaved by Ago2 (Agoshcleave), tailed (AgoshA) and finally trimmed by PARN (Agoshtrim). The resulting single-stranded molecule can dock both 5’ and 3’ ends to facilitate stable loading in Ago2.
Fig 2
Fig 2. In vitro binding of the AgoshRNA-based molecules to Ago2.
The complex forming of hAgo2 and AgoshRNAs was measured by equilibrium fluorescence titration. FAM-labeled substrate (20nM FAM-as2b/s2b) was used as the ds siRNA with known affinity for hAgo2 (black square). Relative fluorescence (y-axis) was scored for all constructs and the x-axis shows the concentration of competitor (AgoshRNAs) added in nM. The graph shows the mean values and standard deviations of two independent experiments.
Fig 3
Fig 3. In vivo processing of the synthetic AgoshRNA molecules.
(A) 100 pmol of the synthetic RNAs was analyzed on a 15% polyacrylamide gel and stained with ethidium bromide. (B) HEK 293T were transfected with 100 pmol of the synthetic RNA molecules. Total cellular RNA was isolated after 48 h and analyzed by northern blot. An LNA probe targeting the 5’ side of the hairpin was used. A size marker is included on the right hand side. Ethidium bromide staining of 5.8S rRNA and tRNAs is included below the blot as loading control. (C) As B, but now using an LNA probe targeting the 3’ side of the hairpin.
Fig 4
Fig 4. Reporter knock down activity of the AgoshRNA molecules.
The knockdown activity of the guide strand of the synthetic RNA molecules was determined by co-transfection with the corresponding luciferase reporter in HEK 293T cells. An unrelated shRNA (shRT5) and the ‘empty’ vector pBluescript served as negative control. We performed three independent transfections, each in duplicate, and standard deviations were calculated.
Fig 5
Fig 5. In vivo complex formation of the AgoshRNA set and Ago2.
(A) Northern blot analysis of immunoprecipitated AgoshRNAs with Ago2-FLAG. Both bound and depleted (unbound) fractions are shown for all synthetic RNA molecules. A size marker is included on the left hand side. Ethidium bromide staining of 5.8S rRNA and tRNAs was included as loading control below the blots. (B) Western blot analysis of the Ago2-bound and unbound fractions using antibodies against FLAG and α-Actin.
Fig 6
Fig 6. In vivo complex formation of the AgoshRNA set with Ago1-4 proteins.
Northern blot analysis of immunoprecipitated pre-Agosh with all four human Ago proteins. Both the bound and depleted (unbound) fractions are shown. A size marker is included on the right hand side. Ago2* was expressed from a different vector as the other four Argonaute proteins. Fold enrichment of bound over unbound is indicated underneath lane. Ethidium bromide staining of 5.8S rRNA and tRNAs is shown as loading control below the blot.
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Grants and funding

This work was supported by the Netherlands Organisation for Scientific Research (Chemical Sciences Division; NWO-CW; Top grant). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.