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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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Cell cycle defects induced by dsRNA. (A) Examples of cell cycle profile phenotypes. Each panel shows the cell cycle profile for Drosophila S2R+ cells that have been treated with the indicated dsRNA. The location of cell populations with G1 and G2/M DNA content is labeled. Cell populations with >G2/M or subG1 DNA content are indicated in panels where there is a significant increase in these cell populations. The number of dsRNAs that displayed an increase of greater than 3 standard deviations from the mean is shown below each phenotype (# of hits). (B) dsRNA targeting cell cycle baits and their interactors cause strong cell cycle defects more frequently than dsRNA targeting other proteins. The percentage of cells in each cell cycle phase following treatment with individual dsRNAs was determined and the number of standard deviations that this value differs from the mean was plotted. Dot plots show data for dsRNAs targeting baits (red diamonds), interactors of the baits (blue squares), non-interactors (green triangles), and GFP (black circles). The red horizontal line in each panel is drawn at 3 standard deviations from the normalized mean of control dsRNA. The genes above the line were considered as hits, or potential cell cycle regulators.
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Figure 2: Cell cycle defects induced by dsRNA. (A) Examples of cell cycle profile phenotypes. Each panel shows the cell cycle profile for Drosophila S2R+ cells that have been treated with the indicated dsRNA. The location of cell populations with G1 and G2/M DNA content is labeled. Cell populations with >G2/M or subG1 DNA content are indicated in panels where there is a significant increase in these cell populations. The number of dsRNAs that displayed an increase of greater than 3 standard deviations from the mean is shown below each phenotype (# of hits). (B) dsRNA targeting cell cycle baits and their interactors cause strong cell cycle defects more frequently than dsRNA targeting other proteins. The percentage of cells in each cell cycle phase following treatment with individual dsRNAs was determined and the number of standard deviations that this value differs from the mean was plotted. Dot plots show data for dsRNAs targeting baits (red diamonds), interactors of the baits (blue squares), non-interactors (green triangles), and GFP (black circles). The red horizontal line in each panel is drawn at 3 standard deviations from the normalized mean of control dsRNA. The genes above the line were considered as hits, or potential cell cycle regulators.

Mentions: We used a previously described library of dsDNA templates [3,65] that allowed for the generation of dsRNAs targeting 596 of the 642 bait protein genes and 1,612 of the 1,843 interaction partner genes. We also generated dsRNAs targeting a random set of 550 genes encoding proteins that were not known to interact with the cell cycle baits or their interactors (Methods). We treated Drosophila S2R+ cells with dsRNAs targeting a total of 2,758 genes and determined cell cycle profiles by flow cytometry. Cell cycle profiles were used to determine the percentage of cells with G1, G2/M, greater than G2/M, or less than G1 DNA content (Additional File 3). dsRNAs that induced a significant increase (>3 standard deviations from the mean) in the percentage of cells in any of these four categories were considered as hits. Examples of cell cycle phenotypes are shown in Figure 2A. Overall, the screen identified 371 unique genes as hits (Additional File 4). A global view of the data reveals that the majority of the strong phenotypes were observed for dsRNAs targeting the putative cell cycle proteins that we used as baits or their interaction partners (Figure 2B). As expected, targeting of the baits resulted in the highest rate (26.0%) of cell cycle defects (Figure 3A). The hit rate for bait interaction partners was 11.8%, significantly higher than the hit rate for the set of random non-interactors (4.5%) (Figure 3A). This was also true for interactors that were derived just from baits that were hits in previous screens without regard to their Gene Ontology annotation. For those, 12.3% (160/1303) were hits, suggesting that prior knowledge of the Gene Ontology annotation of the baits was not necessary to enrich for hits over random genes. Additionally, genes from the group of interactors that were hits interacted with a greater number of baits than did the interactors that were non-hits (p = < .0001) (Additional File 5). We also found that the quality of the protein interaction data affected the hit rate in our RNAi screens. For example, interactors connected to baits by higher confidence interactions [66] were more likely to be hits than those connected by low confidence interactions (Additional File 6). Interestingly, the hit rate for non-interactors was similar to the hit rate observed in undirected genome-wide screens [4,60]. These results show that protein interaction map data can be used to identify a set of genes that is enriched for regulators of a cellular process like the cell cycle. Moreover, the identification of phenotypes for a substantial number of genes that were negative in previous screens shows that the interaction map-guided approach can help to identify putative false negatives from the hits reported in individual screens (e.g., see Figure 3B).


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)

Cell cycle defects induced by dsRNA. (A) Examples of cell cycle profile phenotypes. Each panel shows the cell cycle profile for Drosophila S2R+ cells that have been treated with the indicated dsRNA. The location of cell populations with G1 and G2/M DNA content is labeled. Cell populations with >G2/M or subG1 DNA content are indicated in panels where there is a significant increase in these cell populations. The number of dsRNAs that displayed an increase of greater than 3 standard deviations from the mean is shown below each phenotype (# of hits). (B) dsRNA targeting cell cycle baits and their interactors cause strong cell cycle defects more frequently than dsRNA targeting other proteins. The percentage of cells in each cell cycle phase following treatment with individual dsRNAs was determined and the number of standard deviations that this value differs from the mean was plotted. Dot plots show data for dsRNAs targeting baits (red diamonds), interactors of the baits (blue squares), non-interactors (green triangles), and GFP (black circles). The red horizontal line in each panel is drawn at 3 standard deviations from the normalized mean of control dsRNA. The genes above the line were considered as hits, or potential cell cycle regulators.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Cell cycle defects induced by dsRNA. (A) Examples of cell cycle profile phenotypes. Each panel shows the cell cycle profile for Drosophila S2R+ cells that have been treated with the indicated dsRNA. The location of cell populations with G1 and G2/M DNA content is labeled. Cell populations with >G2/M or subG1 DNA content are indicated in panels where there is a significant increase in these cell populations. The number of dsRNAs that displayed an increase of greater than 3 standard deviations from the mean is shown below each phenotype (# of hits). (B) dsRNA targeting cell cycle baits and their interactors cause strong cell cycle defects more frequently than dsRNA targeting other proteins. The percentage of cells in each cell cycle phase following treatment with individual dsRNAs was determined and the number of standard deviations that this value differs from the mean was plotted. Dot plots show data for dsRNAs targeting baits (red diamonds), interactors of the baits (blue squares), non-interactors (green triangles), and GFP (black circles). The red horizontal line in each panel is drawn at 3 standard deviations from the normalized mean of control dsRNA. The genes above the line were considered as hits, or potential cell cycle regulators.
Mentions: We used a previously described library of dsDNA templates [3,65] that allowed for the generation of dsRNAs targeting 596 of the 642 bait protein genes and 1,612 of the 1,843 interaction partner genes. We also generated dsRNAs targeting a random set of 550 genes encoding proteins that were not known to interact with the cell cycle baits or their interactors (Methods). We treated Drosophila S2R+ cells with dsRNAs targeting a total of 2,758 genes and determined cell cycle profiles by flow cytometry. Cell cycle profiles were used to determine the percentage of cells with G1, G2/M, greater than G2/M, or less than G1 DNA content (Additional File 3). dsRNAs that induced a significant increase (>3 standard deviations from the mean) in the percentage of cells in any of these four categories were considered as hits. Examples of cell cycle phenotypes are shown in Figure 2A. Overall, the screen identified 371 unique genes as hits (Additional File 4). A global view of the data reveals that the majority of the strong phenotypes were observed for dsRNAs targeting the putative cell cycle proteins that we used as baits or their interaction partners (Figure 2B). As expected, targeting of the baits resulted in the highest rate (26.0%) of cell cycle defects (Figure 3A). The hit rate for bait interaction partners was 11.8%, significantly higher than the hit rate for the set of random non-interactors (4.5%) (Figure 3A). This was also true for interactors that were derived just from baits that were hits in previous screens without regard to their Gene Ontology annotation. For those, 12.3% (160/1303) were hits, suggesting that prior knowledge of the Gene Ontology annotation of the baits was not necessary to enrich for hits over random genes. Additionally, genes from the group of interactors that were hits interacted with a greater number of baits than did the interactors that were non-hits (p = < .0001) (Additional File 5). We also found that the quality of the protein interaction data affected the hit rate in our RNAi screens. For example, interactors connected to baits by higher confidence interactions [66] were more likely to be hits than those connected by low confidence interactions (Additional File 6). Interestingly, the hit rate for non-interactors was similar to the hit rate observed in undirected genome-wide screens [4,60]. These results show that protein interaction map data can be used to identify a set of genes that is enriched for regulators of a cellular process like the cell cycle. Moreover, the identification of phenotypes for a substantial number of genes that were negative in previous screens shows that the interaction map-guided approach can help to identify putative false negatives from the hits reported in individual screens (e.g., see Figure 3B).

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