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

Trombly MI, Su H, Wang X - Nucleic Acids Res. (2009)

Bottom Line: This result demonstrates that true RNAi components can be isolated by this screening strategy.Furthermore, Ago2 homozygous mutant ES cells provide a 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

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 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.

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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-TGS 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.
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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-TGS 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.

Mentions: To further examine the effect of the F2V2 mutation on cleavage activity, we attempted genetic rescue of the Ago2 mutant using the Ago2-F2V2 mutant. A BSD-linked HA-tagged Ago2-F2V2 transgene (BSD-HA-Ago2-F2V2) was electroporated into the Ago2 mutant and cells were selected with BSD. For these studies, BSD-linked HA-tagged constructs of the noncleavage competent Argonautes (Ago1, 3 and 4) served as negative controls and BSD-HA-hAgo2 served as a positive control (Figure 6A). The transfected cells were selected with either BSD alone or BSD with 6-TG. Only the hAgo2 construct was capable of rescuing the Ago2 mutant cell line by 6-TG selection (Figure 6A). A western blot for HA confirms that the levels of the BSD-HA-hAgo2 and BSD-HA-hAgo2-F2V2 were comparable, demonstrating that the lack of rescue was not due to a lower level of expression of the hAgo2-F2V2 (Figure 6B). Consistent with drug selection, western blots showed that only Ago2 could repress the Hprt gene through a working RNAi pathway, whereas Ago1, 3, 4 and Ago2-F2V2 were unable to do so (Figure 6B). In addition, we performed an siRNA-based luciferase assay to examine cleavage activity and did not observe any ability of the Ago2-F2V2 mutant protein to repress the luciferase reporter (Figure 6C). Thus, our data are in agreement with Eulalio and colleagues (29), demonstrating that the F2V2 mutations may produce global defects in Ago2's function.Figure 6.


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

Trombly MI, Su H, Wang X - Nucleic Acids Res. (2009)

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-TGS 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.
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Related In: Results  -  Collection

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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-TGS 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.
Mentions: To further examine the effect of the F2V2 mutation on cleavage activity, we attempted genetic rescue of the Ago2 mutant using the Ago2-F2V2 mutant. A BSD-linked HA-tagged Ago2-F2V2 transgene (BSD-HA-Ago2-F2V2) was electroporated into the Ago2 mutant and cells were selected with BSD. For these studies, BSD-linked HA-tagged constructs of the noncleavage competent Argonautes (Ago1, 3 and 4) served as negative controls and BSD-HA-hAgo2 served as a positive control (Figure 6A). The transfected cells were selected with either BSD alone or BSD with 6-TG. Only the hAgo2 construct was capable of rescuing the Ago2 mutant cell line by 6-TG selection (Figure 6A). A western blot for HA confirms that the levels of the BSD-HA-hAgo2 and BSD-HA-hAgo2-F2V2 were comparable, demonstrating that the lack of rescue was not due to a lower level of expression of the hAgo2-F2V2 (Figure 6B). Consistent with drug selection, western blots showed that only Ago2 could repress the Hprt gene through a working RNAi pathway, whereas Ago1, 3, 4 and Ago2-F2V2 were unable to do so (Figure 6B). In addition, we performed an siRNA-based luciferase assay to examine cleavage activity and did not observe any ability of the Ago2-F2V2 mutant protein to repress the luciferase reporter (Figure 6C). Thus, our data are in agreement with Eulalio and colleagues (29), demonstrating that the F2V2 mutations may produce global defects in Ago2's function.Figure 6.

Bottom Line: This result demonstrates that true RNAi components can be isolated by this screening strategy.Furthermore, Ago2 homozygous mutant ES cells provide a 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

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 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.

Show MeSH
Related in: MedlinePlus