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A protein network-guided screen for cell cycle regulators in Drosophila.

Guest ST, Yu J, Liu D, Hines JA, Kashat MA, Finley RL - BMC Syst Biol (2011)

Bottom Line: We demonstrate cell cycle phenotypes for a significant number of these genes and show that the protein interaction network is an efficient predictor of new cell cycle regulators.Our results also show that protein network data can be used to minimize false negatives and false positives and to more efficiently identify comprehensive sets of regulators for a process.Finally, our data provides a high confidence set of genes that are likely to play key roles in regulating the cell cycle or cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, 48201, USA.

ABSTRACT

Background: Large-scale RNAi-based screens are playing a critical role in defining sets of genes that regulate specific cellular processes. Numerous screens have been completed and in some cases more than one screen has examined the same cellular process, enabling a direct comparison of the genes identified in separate screens. Surprisingly, the overlap observed between the results of similar screens is low, suggesting that RNAi screens have relatively high levels of false positives, false negatives, or both.

Results: We re-examined genes that were identified in two previous RNAi-based cell cycle screens to identify potential false positives and false negatives. We were able to confirm many of the originally observed phenotypes and to reveal many likely false positives. To identify potential false negatives from the previous screens, we used protein interaction networks to select genes for re-screening. We demonstrate cell cycle phenotypes for a significant number of these genes and show that the protein interaction network is an efficient predictor of new cell cycle regulators. Combining our results with the results of the previous screens identified a group of validated, high-confidence cell cycle/cell survival regulators. Examination of the subset of genes from this group that regulate the G1/S cell cycle transition revealed the presence of multiple members of three structurally related protein complexes: the eukaryotic translation initiation factor 3 (eIF3) complex, the COP9 signalosome, and the proteasome lid. Using a combinatorial RNAi approach, we show that while all three of these complexes are required for Cdk2/Cyclin E activity, the eIF3 complex is specifically required for some other step that limits the G1/S cell cycle transition.

Conclusions: Our results show that false positives and false negatives each play a significant role in the lack of overlap that is observed between similar large-scale RNAi-based screens. Our results also show that protein network data can be used to minimize false negatives and false positives and to more efficiently identify comprehensive sets of regulators for a process. Finally, our data provides a high confidence set of genes that are likely to play key roles in regulating the cell cycle or cell survival.

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Suppression of G1 arrest by simultaneous knockdown of Dacapo is protein complex-specific. S2R+ cells were treated with dsRNAs targeting the indicated members of the COP9 signalosome, proteasome lid or eIF3 protein complex in combination with either dsRNA targeting GFP (light grey) or Dacapo (dark grey). The percentage of cells with G1 DNA content was determined by flow cytometry. Error bars are standard error of three replicates. Results for additional subunits of these complexes as well as other genes that produced a G1 arrest upon knock down are shown in Additional File 8.
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Figure 5: Suppression of G1 arrest by simultaneous knockdown of Dacapo is protein complex-specific. S2R+ cells were treated with dsRNAs targeting the indicated members of the COP9 signalosome, proteasome lid or eIF3 protein complex in combination with either dsRNA targeting GFP (light grey) or Dacapo (dark grey). The percentage of cells with G1 DNA content was determined by flow cytometry. Error bars are standard error of three replicates. Results for additional subunits of these complexes as well as other genes that produced a G1 arrest upon knock down are shown in Additional File 8.

Mentions: An examination of genes in the list of 210 high confidence cell cycle regulators that displayed a G1 phenotype revealed the presence of multiple members of three structurally related protein complexes: the eukaryotic translation initiation factor 3 complex, the COP9 signalosome, and the proteasome lid. These three complexes have been referred to as "the zomes" based on their related structures. Each complex is composed of protein subunits that contain a domain named PCI (proteasome-COP9-eukaryotic initiation factor) and protein subunits that contain a domain named MPN (Mpr1-Pad1 N-terminus) [84,85]. We sought to identify mechanisms that underlie the G1 phenotype observed following RNAi targeting each of these complexes. Progression of cells through the G1 phase of the cell cycle requires activation of the Cyclin dependent kinase 2 (Cdk2) and Cyclin E (CycE) complex [86-92]. Drosophila Dacapo (Dap), a member of the p21CIP1/p27KIP1 family of Cdk inhibitors, can block progression from G1 into S phase by specifically inhibiting Cdk2/CycE [93,94]. To determine if Dap mediates the G1 arrest induced by RNAi targeting the zomes, cells were treated with dsRNA targeting members of each zome complex alone or in combination with dsRNA targeting the Dap transcript (Figure 5 and Additional File 8). We observed that Dap knockdown completely rescued the G1 arrest induced by RNAi targeting members of the COP9 signalosome, consistent with a similar demonstration by Bjorklund et al. [60] for CSN1b, CSN2 and CSN5. Knockdown of Dap also completely rescued the G1 arrest caused by targeting subunits of the proteasome lid. The COP9 signalosome and proteasome lid complexes both play a role in ubiquitin-mediated proteolysis of Dap/p21/p27 in human and Drosophila cells [95-97]. RNAi targeting the proteasome lid or COP9 signalosome, therefore, likely stabilizes Dap leading to increased Cdk2 inhibition and delayed progression from G1 into S phase. Interestingly, dsRNA targeting Dap transcripts did not have a significant effect on the G1 arrest induced by knocking down members of the eIF3 complex (Figure 5 and Additional File 8). These results suggest that the G1 arrest induced by knocking down eIF3 subunits is not mediated by Dap, or not solely by Dap, and that the underlying mechanism is distinct from that which mediates the COP9 signalosome and proteasome lid phenotypes.


A protein network-guided screen for cell cycle regulators in Drosophila.

Guest ST, Yu J, Liu D, Hines JA, Kashat MA, Finley RL - BMC Syst Biol (2011)

Suppression of G1 arrest by simultaneous knockdown of Dacapo is protein complex-specific. S2R+ cells were treated with dsRNAs targeting the indicated members of the COP9 signalosome, proteasome lid or eIF3 protein complex in combination with either dsRNA targeting GFP (light grey) or Dacapo (dark grey). The percentage of cells with G1 DNA content was determined by flow cytometry. Error bars are standard error of three replicates. Results for additional subunits of these complexes as well as other genes that produced a G1 arrest upon knock down are shown in Additional File 8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113730&req=5

Figure 5: Suppression of G1 arrest by simultaneous knockdown of Dacapo is protein complex-specific. S2R+ cells were treated with dsRNAs targeting the indicated members of the COP9 signalosome, proteasome lid or eIF3 protein complex in combination with either dsRNA targeting GFP (light grey) or Dacapo (dark grey). The percentage of cells with G1 DNA content was determined by flow cytometry. Error bars are standard error of three replicates. Results for additional subunits of these complexes as well as other genes that produced a G1 arrest upon knock down are shown in Additional File 8.
Mentions: An examination of genes in the list of 210 high confidence cell cycle regulators that displayed a G1 phenotype revealed the presence of multiple members of three structurally related protein complexes: the eukaryotic translation initiation factor 3 complex, the COP9 signalosome, and the proteasome lid. These three complexes have been referred to as "the zomes" based on their related structures. Each complex is composed of protein subunits that contain a domain named PCI (proteasome-COP9-eukaryotic initiation factor) and protein subunits that contain a domain named MPN (Mpr1-Pad1 N-terminus) [84,85]. We sought to identify mechanisms that underlie the G1 phenotype observed following RNAi targeting each of these complexes. Progression of cells through the G1 phase of the cell cycle requires activation of the Cyclin dependent kinase 2 (Cdk2) and Cyclin E (CycE) complex [86-92]. Drosophila Dacapo (Dap), a member of the p21CIP1/p27KIP1 family of Cdk inhibitors, can block progression from G1 into S phase by specifically inhibiting Cdk2/CycE [93,94]. To determine if Dap mediates the G1 arrest induced by RNAi targeting the zomes, cells were treated with dsRNA targeting members of each zome complex alone or in combination with dsRNA targeting the Dap transcript (Figure 5 and Additional File 8). We observed that Dap knockdown completely rescued the G1 arrest induced by RNAi targeting members of the COP9 signalosome, consistent with a similar demonstration by Bjorklund et al. [60] for CSN1b, CSN2 and CSN5. Knockdown of Dap also completely rescued the G1 arrest caused by targeting subunits of the proteasome lid. The COP9 signalosome and proteasome lid complexes both play a role in ubiquitin-mediated proteolysis of Dap/p21/p27 in human and Drosophila cells [95-97]. RNAi targeting the proteasome lid or COP9 signalosome, therefore, likely stabilizes Dap leading to increased Cdk2 inhibition and delayed progression from G1 into S phase. Interestingly, dsRNA targeting Dap transcripts did not have a significant effect on the G1 arrest induced by knocking down members of the eIF3 complex (Figure 5 and Additional File 8). These results suggest that the G1 arrest induced by knocking down eIF3 subunits is not mediated by Dap, or not solely by Dap, and that the underlying mechanism is distinct from that which mediates the COP9 signalosome and proteasome lid phenotypes.

Bottom Line: We demonstrate cell cycle phenotypes for a significant number of these genes and show that the protein interaction network is an efficient predictor of new cell cycle regulators.Our results also show that protein network data can be used to minimize false negatives and false positives and to more efficiently identify comprehensive sets of regulators for a process.Finally, our data provides a high confidence set of genes that are likely to play key roles in regulating the cell cycle or cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, 48201, USA.

ABSTRACT

Background: Large-scale RNAi-based screens are playing a critical role in defining sets of genes that regulate specific cellular processes. Numerous screens have been completed and in some cases more than one screen has examined the same cellular process, enabling a direct comparison of the genes identified in separate screens. Surprisingly, the overlap observed between the results of similar screens is low, suggesting that RNAi screens have relatively high levels of false positives, false negatives, or both.

Results: We re-examined genes that were identified in two previous RNAi-based cell cycle screens to identify potential false positives and false negatives. We were able to confirm many of the originally observed phenotypes and to reveal many likely false positives. To identify potential false negatives from the previous screens, we used protein interaction networks to select genes for re-screening. We demonstrate cell cycle phenotypes for a significant number of these genes and show that the protein interaction network is an efficient predictor of new cell cycle regulators. Combining our results with the results of the previous screens identified a group of validated, high-confidence cell cycle/cell survival regulators. Examination of the subset of genes from this group that regulate the G1/S cell cycle transition revealed the presence of multiple members of three structurally related protein complexes: the eukaryotic translation initiation factor 3 (eIF3) complex, the COP9 signalosome, and the proteasome lid. Using a combinatorial RNAi approach, we show that while all three of these complexes are required for Cdk2/Cyclin E activity, the eIF3 complex is specifically required for some other step that limits the G1/S cell cycle transition.

Conclusions: Our results show that false positives and false negatives each play a significant role in the lack of overlap that is observed between similar large-scale RNAi-based screens. Our results also show that protein network data can be used to minimize false negatives and false positives and to more efficiently identify comprehensive sets of regulators for a process. Finally, our data provides a high confidence set of genes that are likely to play key roles in regulating the cell cycle or cell survival.

Show MeSH
Related in: MedlinePlus