Cleavage independent siRNA-mediated degradation of mRNA in the absence of Ago2.

<p>Ago2^fl/fl MEF cells and Ago2^−/− MEF cells were transfected with 10 nM of siRNAs targeting Luciferase, or CDS, or 3′UTR of the Rab5c mRNA. bDNA assay (A) and 5′RACE (B) were performed on total RNA isolated from cells 3 h post-transfection. (A) Data is presented as mean ± s.d. for two technical replicates of bDNA measurement. (B) 5′RACE nested PCR detection of cleavage product. Numbers of clones bearing expected inserts are indicated below corresponding lanes of the gel (expected PCR product length is 145 bp; expected adapter/Rab5c junction sequence is shown below; 16 clones were sequenced for each PCR reaction).</p

siRNAs targeting CDS and 3′UTR differ in their Ago2 dependence <i>in vivo</i>.

<p>Data points are levels of mRNA measured by qRT-PCR in the livers (harvested 24 hours post-injection) of mice i.v.-injected with LNP-formulated siRNAs, as described in Materials and Methods, expressed as a percentage of mRNA levels in control siRNA-treated animals. (A) mRNA expression of Argonautes and Dicer1 in Ago2^fl/fl and Ago2^−/− mouse liver (mean ± s.d., n = 4). (B) Levels of Fads1 mRNA in livers of Ago2^−/− mice and Ago2^fl/fl mice (mean ± s.d., n = 2–3) treated with siRNAs targeting Fads1 CDS or 3′UTR at 0.5 mg/kg. (C) Rab5c mRNA levels after treatment with siRNAs targeting its CDS or 3′UTR-far at 1 mg/kg (mean ± s.d., n = 2–3). (D) FVII mRNA in the liver of animals treated with siRNAs (mean ± s.d., n = 4–5, doses: 0.8 mg/kg for siRNA targeting CDS and 0.4 mg/kg for siRNA targeting 3′UTR) and FVII protein in the serum quantified by chromogenic assay. (E) Activity of siRNAs targeting CDS and 3′UTR in the absence of Ago1, 3, or 4. Legend in the bottom left corner of the graph also indicates the line in the X-axis describing the treatment type by gene (mean ± s.d., n = 3). Mice knockout for different individual Argonaute genes and C57BL/6 control animals were i.v.-injected with one of two combinations of LNP-formulated siRNAs targeting Fads1 and Rab5c (CDS-targeting for one gene, at 0.8 mg/kg and 3′UTR-targeting for the other gene, at 0.4 mg/kg) or with control Luciferase siRNA at 1.2 mg/kg.</p

Effect of individual Argonautes in vivo.

<p>(A) C57BL/6 mice were i.v.-injected with LNP-formulated siRNA targeting Ago2–3′UTR or with control Luciferase siRNA at 0.5 mg/kg. Animals were sacrificed at indicated time points and Ago2 mRNA was quantified by bDNA assay (mean ± s.d., n = 5). (B) bDNA measurement of mRNA knockdown of Rab5c (by siRNA targeting 3′UTR-near at 0.5 mg/kg) and FVII (by siRNA targeting CDS at 0.2 mg/kg) in wild-type mice (mean ± s.d., n = 4–5) following 4 days of Ago2 knockdown (by siRNA targeting 3′UTR at 0.5 mg/kg). (C) Animals were i.v.-injected with Ago1, Ago2, or Luc siRNA on day 1, Ago1 treated animals were further i.v.-injected with Ago2 on day 2; and Rab5c-3′UTR-near siRNA was introduced on day 5 (where indicated; each siRNA was dosed at 0.5 mg/kg). All animals were sacrificed on day 6. Liver mRNA levels for Ago1, Ago2, and Rab5c were measured by bDNA assay (mean ± s.d., n = 4–5). (D) Mice were i.v.-injected with LNP containing 0.5 mg/kg siRNA against each of Ago1 and Ago2 (or 1 mg/kg of Luciferase control siRNA) twice at week long intervals, at the end of the second week mice were i.v.-injected with Fads1–3′UTR or Luciferase control siRNAs at 0.4 mg/kg. Mice were sacrificed for liver RNA extraction the day after the last injection (mean ± s.d., n = 3).</p

Differential Ago2 dependence of siRNAs targeting coding sequence (CDS) and 3′-untranslated region (3′UTR) of mRNA <i>in vitro</i>.

<p>(A) Mouse embryonic fibroblast (MEF) cells, Ago2^fl/fl and Ago2^−/− (wild-type and Ago2 knockout) were transfected with 10 nM of siRNAs targeting CDS or 3′UTR (16 or 28 siRNAs, respectively, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101749#pone.0101749.s004" target="_blank">Table S1</a>) of mRNAs of four genes: Fads1, Fads2, Rab5a, and Rab5c. Levels of expression of target-gene mRNA in Ago2^fl/fl MEF cells and Ago2^−/− MEF cells measured by branched DNA (bDNA) assay 24 h post-transfection were plotted as percentage of relative target mRNA compared to Luciferase siRNA transfected controls. (B, C) Ago2^−/− MEF cells (square) or Ago2^fl/fl MEF cells (circle) were transfected with siRNAs targeting CDS or 3′UTR of Fads1 (B) or Rab5c (C) mRNA (relative positions of target sites within mRNA are shown). Transfection and assay as described in A, but dilution series with the maximum dose of 40 nM were done. Combined results of two independent transfections are shown. (D) Ago2^fl/fl MEF cells were transfected with 10 nM siRNA targeting 3′UTR of Ago2 (Eif2c2) mRNA or Luciferase, followed by transfection with two combinations of siRNAs targeting Fads1 and Rab5c (CDS-targeting for one gene and 3′UTR-targeting for the other gene, 10 nM each, or 20 nM of Luciferase siRNA control) 48 hours later. Levels of target-genes and Ago2 mRNA expression measured by bDNA assay 24 h after second transfection were plotted as percentage of relative target mRNA compared to Luciferase siRNA-transfected control (mean ± s.d., n = 3). Legend in the bottom left corner of the graph also indicates the line in the X-axis describing the treatment type (none, CDS, 3′UTR) by target gene.</p