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A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.

Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID - BMC Mol. Biol. (2007)

Bottom Line: Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors.Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. xiaocui_zhu@hotmail.com

ABSTRACT

Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.

Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.

Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

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Efficient knockdown of both PKA alpha and beta catalytic subunits is necessary to attenuate PKA-dependent phosphorylation and transcription. (a) Phosphorylation of the cytoskeletal protein VASP in response to cAMP analogs; combined knockdown of Pka Cα and Cβ is required to attenuate phosphorylation in response to either 8Br-cAMP or 8pCPT-cAMP. (b-e) Induction of PKA-dependent transcripts Nr4a1, Nr4a2, Ctla2α and Ctla2β in response to 8pCPT-cAMP. Induction of all 4 transcripts is significantly attenuated only upon depletion of both Pka Cα and Cβ (Data represent averages (± SEM) from 2 independent experiments).
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Figure 4: Efficient knockdown of both PKA alpha and beta catalytic subunits is necessary to attenuate PKA-dependent phosphorylation and transcription. (a) Phosphorylation of the cytoskeletal protein VASP in response to cAMP analogs; combined knockdown of Pka Cα and Cβ is required to attenuate phosphorylation in response to either 8Br-cAMP or 8pCPT-cAMP. (b-e) Induction of PKA-dependent transcripts Nr4a1, Nr4a2, Ctla2α and Ctla2β in response to 8pCPT-cAMP. Induction of all 4 transcripts is significantly attenuated only upon depletion of both Pka Cα and Cβ (Data represent averages (± SEM) from 2 independent experiments).

Mentions: Mammalian cells express two Pka catalytic subunits, Cα and Cβ. Mice are viable after knockout of either gene, suggesting that either isoform can perform the required physiological functions of this kinase [15,16]. To determine the functional consequences of depleting both of the Pka C subunits in RAW cells, we assessed the cAMP-induced phosphorylation of the vasodilator stimulated phosphoproteins (VASP), a known substrate of Pka [17]. In response to cAMP analogs, we observed a significant increase in phospho-VASP, which was unaffected in cells depleted of either Pka Cα or Pka Cβ (Fig. 4a). However, in cells with both Cα and Cβ knocked down, there was almost complete attenuation of VASP phosphorylation in response to cAMP (Fig. 4a). It is well established that Pka mediates many transcriptional effects, primarily through phosphorylation and activation of cAMP response element (CRE) binding protein (CREB) and cAMP response element modulator (CREM) [18]. Based on a recent microarray study in RAW cells [19], we identified several cAMP-dependent mRNAs and selected four targets for assessment in our Pka knockdown cell lines; Nr4a1, Nr4a2, Ctla2α and Ctla2β. Nr4a1/Nur77 and Nr4a2/Nurr1, members of the NR4A subfamily of orphan nuclear receptors, are potential transcriptional mediators in many cell types and have been shown to be regulated by cAMP [20]. Regulation of the transcription of Ctla2α and Ctla2β, cysteine protease inhibitors originally identified in cytotoxic T lymphocytes [21], has not been reported previously. None of these cAMP-dependent transcripts show any significant alteration of 8pCPT-cAMP induced increase in cells depleted for either Pka catalytic subunit, whereas depletion of both Pka Cα and Cβ leads to a marked decrease in transcript induction (Fig. 4b–e). These data provide the first evidence that Ctla2 transcription is PKA-dependent. More importantly, our data demonstrate the value of an RNAi platform which permits the depletion of multiple endogenous gene targets from a single viral infection of an intractable cell line. The system allows the demonstration of downstream biological consequences of the loss of a function redundantly performed by related proteins. Only upon effective depletion of both Pka C subunits is a phenotype observed (Fig. 4). There are numerous examples, especially in G-protein mediated signaling pathways, where mammalian cells express multiple proteins with redundant function, presumably to build robustness into the system. It is therefore noteworthy that the cells maintain a small degree of response in the recorded readouts in the dual knockdown cells (Fig. 4). Although this result could argue that an alternative cAMP effector, such as Epac, could contribute, it seems more likely that the residual Pka catalytic subunit (Fig. 3d+e) is capable of mediating the residual response. This observation supports the inherent robustness of this signaling pathway, and is consistent with the finding that the Pka Cα knockout mice remain viable despite certain cell types showing <10% of the wild type level of total PKA activity [16].


A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.

Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID - BMC Mol. Biol. (2007)

Efficient knockdown of both PKA alpha and beta catalytic subunits is necessary to attenuate PKA-dependent phosphorylation and transcription. (a) Phosphorylation of the cytoskeletal protein VASP in response to cAMP analogs; combined knockdown of Pka Cα and Cβ is required to attenuate phosphorylation in response to either 8Br-cAMP or 8pCPT-cAMP. (b-e) Induction of PKA-dependent transcripts Nr4a1, Nr4a2, Ctla2α and Ctla2β in response to 8pCPT-cAMP. Induction of all 4 transcripts is significantly attenuated only upon depletion of both Pka Cα and Cβ (Data represent averages (± SEM) from 2 independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Efficient knockdown of both PKA alpha and beta catalytic subunits is necessary to attenuate PKA-dependent phosphorylation and transcription. (a) Phosphorylation of the cytoskeletal protein VASP in response to cAMP analogs; combined knockdown of Pka Cα and Cβ is required to attenuate phosphorylation in response to either 8Br-cAMP or 8pCPT-cAMP. (b-e) Induction of PKA-dependent transcripts Nr4a1, Nr4a2, Ctla2α and Ctla2β in response to 8pCPT-cAMP. Induction of all 4 transcripts is significantly attenuated only upon depletion of both Pka Cα and Cβ (Data represent averages (± SEM) from 2 independent experiments).
Mentions: Mammalian cells express two Pka catalytic subunits, Cα and Cβ. Mice are viable after knockout of either gene, suggesting that either isoform can perform the required physiological functions of this kinase [15,16]. To determine the functional consequences of depleting both of the Pka C subunits in RAW cells, we assessed the cAMP-induced phosphorylation of the vasodilator stimulated phosphoproteins (VASP), a known substrate of Pka [17]. In response to cAMP analogs, we observed a significant increase in phospho-VASP, which was unaffected in cells depleted of either Pka Cα or Pka Cβ (Fig. 4a). However, in cells with both Cα and Cβ knocked down, there was almost complete attenuation of VASP phosphorylation in response to cAMP (Fig. 4a). It is well established that Pka mediates many transcriptional effects, primarily through phosphorylation and activation of cAMP response element (CRE) binding protein (CREB) and cAMP response element modulator (CREM) [18]. Based on a recent microarray study in RAW cells [19], we identified several cAMP-dependent mRNAs and selected four targets for assessment in our Pka knockdown cell lines; Nr4a1, Nr4a2, Ctla2α and Ctla2β. Nr4a1/Nur77 and Nr4a2/Nurr1, members of the NR4A subfamily of orphan nuclear receptors, are potential transcriptional mediators in many cell types and have been shown to be regulated by cAMP [20]. Regulation of the transcription of Ctla2α and Ctla2β, cysteine protease inhibitors originally identified in cytotoxic T lymphocytes [21], has not been reported previously. None of these cAMP-dependent transcripts show any significant alteration of 8pCPT-cAMP induced increase in cells depleted for either Pka catalytic subunit, whereas depletion of both Pka Cα and Cβ leads to a marked decrease in transcript induction (Fig. 4b–e). These data provide the first evidence that Ctla2 transcription is PKA-dependent. More importantly, our data demonstrate the value of an RNAi platform which permits the depletion of multiple endogenous gene targets from a single viral infection of an intractable cell line. The system allows the demonstration of downstream biological consequences of the loss of a function redundantly performed by related proteins. Only upon effective depletion of both Pka C subunits is a phenotype observed (Fig. 4). There are numerous examples, especially in G-protein mediated signaling pathways, where mammalian cells express multiple proteins with redundant function, presumably to build robustness into the system. It is therefore noteworthy that the cells maintain a small degree of response in the recorded readouts in the dual knockdown cells (Fig. 4). Although this result could argue that an alternative cAMP effector, such as Epac, could contribute, it seems more likely that the residual Pka catalytic subunit (Fig. 3d+e) is capable of mediating the residual response. This observation supports the inherent robustness of this signaling pathway, and is consistent with the finding that the Pka Cα knockout mice remain viable despite certain cell types showing <10% of the wild type level of total PKA activity [16].

Bottom Line: Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors.Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. xiaocui_zhu@hotmail.com

ABSTRACT

Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.

Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.

Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

Show MeSH
Related in: MedlinePlus