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PMID:19528325

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Citation

Dorfman, M, Gomes, JE, O'Rourke, S and Bowerman, B (2009) Using RNA interference to identify specific modifiers of a temperature-sensitive, embryonic-lethal mutation in the Caenorhabditis elegans ubiquitin-like Nedd8 protein modification pathway E1-activating gene rfl-1. Genetics 182:1035-49

Abstract

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.

Links

PubMed PMC2728846 Online version:10.1534/genetics.109.104885

Keywords

Animals; Caenorhabditis elegans/embryology; Caenorhabditis elegans/genetics; Caenorhabditis elegans Proteins/metabolism; Embryo, Nonmammalian/cytology; Embryonic Development; Genes, Lethal; Mutation; RNA Interference; Temperature; Ubiquitin-Activating Enzymes/metabolism; Ubiquitins/genetics; Ubiquitins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:SAO1

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:SAO1

located_in

GO:0032154: cleavage furrow

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:SAO1

located_in

GO:0005938: cell cortex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:SAO1

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:G3MU54

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:G5ECX9

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:O17838

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O44136

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O44490

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:O45394

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O45394

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O45394

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:O45828

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O45828

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:O45828

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:CSN5

involved_in

GO:0000338: protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:UBA3

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:UBA3

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:UBA3

involved_in

GO:0045116: protein neddylation

ECO:0000315: mutant phenotype evidence used in manual assertion

WB:WBVar00094698

P

Seeded From UniProt

complete

CAEEL:MNK

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:MNK

involved_in

GO:0060625: regulation of protein deneddylation

ECO:0000316: genetic interaction evidence used in manual assertion

WB:WBGene00004341

P

Seeded From UniProt

complete

CAEEL:SEL10

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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