The Caenorhabditis elegans cullin-RING ubiquitin ligase CRL4DCAF-1 is required for proper germline nucleolus morphology and male development

Abstract

Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in Caenorhabditis elegans that is marked by abnormal globular morphology of the nucleolus and fewer germ cells. We identified DDB1 Cullin4 associated factor (DCAF)-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, the inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of the germ line-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor periodic trptophan protein 1 (PWP1). We observed that the inactivation of Caenorhabditis elegansDCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.

Keywords: DCAF1; germ cell; male lethality; male tail; nucleolus; ribosome biogenesis.

Fig. 1.

dcaf-1 inactivation phenocopies the ddb-1 mutant germline nucleolus defect. a) DIC images of one gonad arm for wild type and ddb-1(tm1769) mutant. The boxed region in the upper image is shown at higher magnification in the bottom image. b) DIC images of germ cells in control RNAi, ZK1259.1 (dcaf-1) RNAi, dcaf-1(ok1867), and ncl-2(e1896). Note that the 3 methods of inactivating dcaf-1 phenocopy the ddb-1 mutant germ cell nucleolus morphology defect. Scale bars, 10 µm. c) Schematic of the dcaf-1 genomic locus (exons are boxes and solid lines are introns). The region deleted in the dcaf-1(ok1867) mutant is between the dotted lines. The location of the dcaf-1(e1896) missense mutation that gives rise to the E445K protein change is marked. d) An alignment of the region flanking the E445K mutation (marked by an asterisk) in allele e1896 for orthologs in several diverse animal species (Asterias rubens, Danio rerio, Gallus domesticus, and Homo sapiens). Residues that are conserved in all of the species are shaded.

Fig. 2.

Transmission electron micrographs of dcaf-1 mutant germ cells and hypodermal cells. a–d) Micrographs of wild type and dcaf-1(e1896) mutant germ cells in the distal region of the gonad. Panels b′ and d′) show the larger-boxed regions in panels B and D at higher magnification. Nu, nucleolus; N, nucleus; C, cytoplasm. e and f) Micrographs of wild type and dcaf-1(e1896) mutant hypodermal cells in the Hyp7 syncytium. Panels b″, d″, e″, and f″) show the smaller-boxed regions of panels b, d, e, and f) at higher magnification. g) Graph of the number of ribosomes per arbitrary area in germ cells and hypodermis (Hyp7) of wild type and dcaf-1(e1896) mutants; n = 20 counts for each condition except for germ cells in dcaf-1(e1896) mutants that had 40 counts. ns, not significant; ****P-value <0.0001. Statistics are comparisons to the appropriate wild-type control. Throughout, error bars are SEM.

Fig. 3.

dcaf-1 and ddb-1 mutant male tail and germ cell phenotypes. a) DIC micrographs of the distal end of the adult male germ line for wild type, dcaf-1(ok1867) mutant, and ddb-1(tm1769) mutant. The region with the distal end is denoted by an asterisk outside the body. b) DIC micrographs of the adult male tail for wild type, dcaf-1 mutant, and ddb-1 mutant. Sensory rays and fan are marked on the wild-type image (not all of the 9 rays per side are in focus in the DIC image). Rays and fans are not fully developed on dcaf-1 and ddb-1 mutant male tails. Scale bars, 10 µm.

Fig. 4.

FOG-1 inactivation rescues the dcaf-1 germ cell defect and FOG-1 and FOG-3 levels increase in adult dcaf-1(RNAi) germ lines. a) DIC images of the germ line of fog-1(q253ts) mutant adults treated with control, dcaf-1, and ddb-1 RNAi, and grown at 25°C. The distal region of the gonad is to the top left (marked by an asterisk). b) Immunofluorescence of dissected gonads stained with Hoechst 33258 DNA stain and anti-Myc and anti-FLAG antibodies in adults expressing 3xMyc-FOG-1 and FOG-3::3xFLAG treated with control RNAi (top) or dcaf-1 RNAi (middle). Wild-type adults treated with dcaf-1 RNAi (bottom) were used as a negative control for Myc and FLAG staining. The distal tip of the gonads is to the left (marked by an asterisk). Scale bars, 10 µm. c) Graph of the level of 3xMyc::FOG-1 and FOG-3::3xFLAG in the distal and proximal regions of the germ line of adults treated with control or dcaf-1 RNAi.

Fig. 5.

PWP-1 levels are negatively regulated by CRL4^DCAF-1. a) wrmScarlet::PWP-1 levels in L4-stage larvae treated with control and dcaf-1 RNAi from hatch. Note the increased levels of wrmScarlet::PWP-1 in the nucleoli of intestine cells and germ cells with dcaf-1 RNAi. Arrowheads mark the anterior end of the animal. b) wrmScarlet::PWP-1 levels in gonad arms of L4-stage larvae treated with control and dcaf-1 RNAi from hatch. c) wrmScarlet::PWP-1 levels in gonad arms of adults treated with control and dcaf-1 RNAi for 3 days from the L3 larval stage. Asterisks mark the distal end of the germ line. Scale bars, 10 µm. d) Graph of wrmScarlet::PWP-1 levels in the intestine of wild type and fog-1(q253ts) mutant L4-stage larvae grown from hatching with control and dcaf-1 RNAi. The level of wild-type larvae treated with control RNAi is set to 100. e) Graph of wrmScarlet::PWP-1 levels in the distal and proximal germ line of adult wild type and fog-1(q253ts) mutants were grown with control and dcaf-1 RNAi from the L3-stage at 25°C for 3 days. The distal wild-type control RNAi level is set to 100. ns, not significant; *P-value < 0.05; ****P-value <0.0001. Statistics without lines refer to comparisons with the appropriate control RNAi.

Fig. 6.

PWP-1 inactivation rescues the dcaf-1 RNAi germline nucleolus morphology defect but not the developmental delay, which is rescued by fog-1 inactivation. a) Wild-type and homozygous pwp-1(ok3322) L2/L3-stage larvae treated with dcaf-1 RNAi. The ventral side of the larvae is down. Note that wild type treated with dcaf-1 RNAi has the germline nucleolus defect, with some cells that have normal morphology being somatic gonadal cells. The pwp-1 homozygote treated with dcaf-1 RNAi has normal nucleolus morphology in germ cells. b) pwp-1(ok3322)/nT1 heterozygote L4-stage larvae grown on dcaf-1 feeding-RNAi bacteria. Note the normal germ cell nucleolus morphology. Asterisks mark the distal end of the germ line. Scale bars, 10 µm. c) Graph of the timing of larval development from hatch to adult at 25°C for wild type, pwp-1(ok3322)/nT1 heterozygotes, and fog-1(q253ts). Note that dcaf-1 RNAi treatment induces a larval developmental delay in wild-type animals and heterozygous pwp-1/nT1 mutants, but not in fog-1(q253ts) mutants.