C. elegans dss-1 is functionally conserved and required for oogenesis and larval growth

Abstract

Background: Dss1 (or Rpn15) is a recently identified subunit of the 26S proteasome regulatory particle. In addition to its function in the protein degradation machinery, it has been linked to BRCA2 (breast cancer susceptibility gene 2 product) and homologous DNA recombination, mRNA export, and exocytosis. While the fungal orthologues of Dss1 are not essential for viability, the significance of Dss1 in metazoans has remained unknown due to a lack of knockout animal models.

Results: In the current study deletion of dss-1 was studied in Caenorhabditis elegans with a dss-1 loss-of-function mutant and dss-1 directed RNAi. The analysis revealed an essential role for dss-1 in oogenesis. In addition, dss-1 RNAi caused embryonic lethality and larval arrest, presumably due to loss of the dss-1 mRNA maternal contribution. DSS-1::GFP fusion protein localised primarily in the nucleus. No apparent effect on proteasome function was found in dss-1 RNAi treated worms. However, expression of the C. elegans dss-1 in yeast cells deleted for its orthologue SEM1 rescued their temperature-sensitive growth phenotype, and partially rescued the accumulation of polyubiquitinated proteins in these cells.

Conclusion: The first knockout animal model for the gene encoding the proteasome subunit DSS-1/Rpn15/Sem1 is characterised in this study. In contrast to unicellular eukaryotes, the C. elegans dss-1 encodes an essential protein, which is required for embryogenesis, larval growth, and oogenesis, and which is functionally conserved with its yeast and human homologues.

Figure 1

Expression of C. elegans dss-1. A. dss-1 mRNA is expressed at different larval ages (L1 – L4) and in adult worms (ya, young adult) as shown by RT-PCR. act-4, actin (M03F4.2). B. DSS-1::GFP expressed under the dss-1 promoter localises to the nucleus of intestinal cells as shown by colocalisation with DAPI. g, gonad; i, intestine.

Figure 2

dss-1 deletion results in defects in oogenesis and intestinal absorption. A. dss-1 gene is disrupted in the tm370 allele by a deletion spanning exon 2. B. dss-1(tm370) adults are sterile and thinner than control heterozygotes. Oocytes (C) and embryos (F) are visible in a control dss-1 heterozygote but dss-1 homozygotes have no (D) or only a few oocytes (E). G. Structures resembling embryos are occasionally seen in the uterus of dss-1 homozygotes. Asterisk, spermatheca; e, embryo or embryo-like structure; g, gonad; i, intestine; o, oocyte; u, uterus. H. The average number of oocytes in dss-1 homozygotes is reduced. Oocytes were identified either by Nomarski optics or by DAPI staining. n, number of individual gonad arms examined; ND, not determined. I. Spermatocytes (arrow) are still present in a dss-1 homozygote. I'. DAPI staining. Asterisk marks the more differentiated spermatids. J. Intestinal epithelial cell nuclei can be visualized by DAPI staining in a control N2 L4 worm. K. In dss-1 homozygotes an expanded intestinal lumen filled with bacteria can be seen. L. The pharynx is often twisted, possibly by pressure from the enlarged intestine.

Figure 3

dss-1 RNAi results in sterility and growth defects. A. dss-1 RNAi treated worms have higher embryonic lethality and larval arrest compared to vector-treated control worms. One representative of three separate 26°C RNAi experiments is plotted. F1 progeny were followed for four days after N2 L4 worms were placed on RNAi plates. B. Vector-treated control N2 worms have a normal number of oocytes, but in dss-1 RNAi treated worms the oocytes are absent (C) or present in reduced numbers (D). Embryo-like structures in the uterus (E, arrow), vacuoles (F, arrowheads), and emo cells (G, G', em) in the gonad are found occasionally. DTC cells are present in both vector- (H, H') and dss-1 RNAi (I. I') treated worms. Deformed tail (J) and head epithelia (K) are seen in worms arrested at early larval stages. L. Undigested bacteria are found in the bloated intestinal lumen.

Figure 4

dss-1 phenotype resembles both brc-2 and kgb-1 mutants. A. brc-2(tm1086) mutants are sterile with a uterus filled with abnormal embryos. B. Abnormal diakinesis occurs in brc-2 oocytes as shown by DAPI staining. C. dss-1 oocytes appear to have the expected number of six condensed chromosomes, but they occasionally show defects in diakinesis (D). In B-D only one focal plane is shown. E-M. Nomarski optics; E'-M' DAPI staining. E. kgb-1 mutants grown at 26°C lack embryos in the uterus (u). Their gonads are often disorganised (F, F', arrows indicate oocyte chromosomes) and similar disorganisation is sometimes seen in dss-1 homozygotes (G, G', arrow indicates an oocyte). DAPI-positive emo oocytes are often present in kgb-1 (H), and occasionally in dss-1 gonads (I). J. emo-like structures are also seen in dss-1 uteri. K-M. rpn-10 mutants grown at the restrictive temperature (25°C) have oocytes (K) but lack sperm (L, M). 15°C, permissive temperature. Asterisk, spermatheca; em, emo cell; g, germ cells; u, uterus; v, vulva.

Figure 5

C. elegans dss-1 can partially rescue yeast sem1 mutation. A. S. cerevisiae sem1 mutants have a growth defect at restrictive temperature (vector). Expression of either C. elegans dss-1 or the yeast SEM1 in sem1 mutants rescues the growth defect. B. Accumulation of polyubiquitinated proteins is seen in sem1 mutants transformed with an empty vector both at permissive and restrictive temperature at two different time points. Expression of yeast SEM1 in sem1 mutant cells reduces efficiently the accumulation of polyubiquitinated proteins, whereas expression of C. elegans dss-1 has only a modest effect. Lower panels have been blotted with anti-Sso1/2 antibody as a control for equal loading.