A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells

Abstract

Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

Figure 1.

A recessive genetic screen to isolate RNAi mutants in Blm-deficient ES cells. The screen is diagrammed on the left and the drug resistance phenotype of the cells is shown on the right. Viable ES cells appear dark blue when stained with methylene blue. (A) Blm^−/− Hprt^+/+ cells are HAT^R and 6-TG^S. (B) After the addition of a puromycin-linked U6-shRNA-Hprt to silence the Hprt gene, cells become HAT^S and 6-TG^R. (C) Gene trap mutagenesis generates mutations in the cells and can be selected with the drug G418. (D) The Blm-deficient gene trapped ES cells accumulate homozygous gene trap mutations. Mutations that inactivate the RNAi pathway lead to Hprt expression and these cells are HAT^R and Puro^R.

Figure 2.

Diagram of retroviral gene trap constructs. (A) Schematic of how gene traps create mutations after integration. As an example, integration of a proviral gene trap between exons 1 and 2 leads to loss of exons 2 and 3. (B) Diagrams of PGGV2-PGGV7 are depicted. Grey bars in PGGV5-7 represent unique insertion in LTR. The titer for each construct is shown to the right. SA, splice acceptor; LacZ, beta-galactosidase; Neo, neomycin resistance marker; PolA, polyadenylation.

Figure 3.

Confirmation of RNAi mutants. (A) Methylene blue staining of viable cells showing the drug resistance of a putative RNAi mutant as compared to the reporter cell line, 59Z4. (B) A Southern blot of EcoRI digested DNA using a probe unique to the gene trap identifies distinct gene-trap integration events by size. (C) Western and northern blots show that Hprt protein and RNA expression returns to levels similar to the parental cell line, PGG5-4. For the western blot, β-tubulin is shown as a loading control. For the northern blot, the 28S and 18S rRNAs are shown as a loading control. (D) A luciferase assay was used to confirm that HAT^R clones are RNAi-defective. The fold repression of F-luc by the luc1-shRNA is shown for the reporter (59Z4) and mutant cell lines. The data shown are the average of six independent luciferase assays, and error bars represent the standard error of the mean. The F-luc data was normalized to R-luc and the fold repression of F-luc by the control shRNA was set to 1 (not plotted). The reporter cell line represses the F-luc reporter 35-fold while the mutants repress 1- to 2-fold.

Figure 4.

Isolation of Ago2 mutants. (A) All Ago2 gene-trap mutants were identified in the first intron of the Ago2 locus, six are depicted in the diagram with the gene trap construct that was used in the screen shown above the arrowheads. A western blot for Ago2 shows that endogenous Ago2 was below detection in the mutant cells. (B) To confirm that the isolated Ago2 mutant was homozygous for the gene trap, a Southern blot was performed on EcoRI digested DNA from the reporter cell line and one of the Ago2 mutants, Ago2# 13. A probe for a region flanking the Ago2 gene trap was used to detect the wild-type locus as a 3.1 kb fragment and the gene trap locus as a 5.7 kb fragment. The Ago2 mutant is homozygous for the gene trap as shown by the detection of only the 5.7 kb fragment.

Figure 5.

Rescue of the gene trap Ago2 mutant by a hAgo2 transgene. (A) Schematic of the hAgo2 rescue construct. (B) Rescue of the Ago2 mutant reverts the drug resistance phenotype to HAT^S and 6-TG^R (compare to the drug resistance phenotype of the Ago2 mutant). (C) Rescue of the Ago2 mutant also reverts the levels of Hprt back to reporter cell (59Z4) levels as shown by northern and western blots. An Ago2 western blot performed on parental (PGG5-4), reporter (59Z4), mutant and rescued cell lines showing similar levels of Ago2 in the rescued cells. (D) A luciferase assay for RNAi-directed mRNA cleavage confirms the rescue of Ago2 mutant by hAgo2 transgene. (E) A luciferase assay was used to examine miRNA repression and shows that the Ago2 mutant exhibits a slight decrease in miRNA repression compared to the original reporter cell line 59Z4. Calculations for data shown in (D) and (E) were performed as in Figure 3D.

Figure 6.

Ago2 mutant ES cells can serve as a tool to perform mutational analysis. (A) The mutant Ago2 cell line was transfected with transgenes expressing a BSD-linked, HA-tagged cDNA construct for Ago1, Ago2, Ago3, Ago4 and Ago2-F2V2. Cells were selected with BSD or BSD with 6-TG. Only stable transfection of WT-Ago2 can revert the 6-TG^S phenotype of the mutant cells. (B) Western blot for HA shows the expression levels of different Ago proteins. Western blot for Hprt shows that only Ago2 expression silences Hprt expression. Tubulin is shown as a loading control. (C) A luciferase assay for RNAi-directed cleavage activity shows that Ago2-F2V2 is deficient for cleavage activity. (D) Coimmunoprecipitation performed in 293T cells shows that GFP-Ago2 is associated with Flag-tagged Ago2 as well as Flag-tagged Ago2-F2V2.