The Caenorhabditis elegans proteasome subunit RPN‐12 is required for hermaphrodite germline sex determination and oocyte quality

Abstract

Background The proteasome is a multi‐subunit complex and a major proteolytic machinery in cells. Most subunits are essential for proteasome function, and depletion of individual subunits normally results in lethality. RPN‐12/Rpn12/PSMD8 is a lid subunit of the 19S regulatory particle (RP) of the 26S proteasome. Studies in Caenorhabditis elegans demonstrated that RNAi depletion of RPN‐12 does not result in lethality. RPN‐12 has not been well studied in higher eukaryotes. In this study, we investigate the biological significance of RPN‐12 in C. elegans. Results We found that the null mutant rpn‐12(av93) did not cause major impairment of the proteolytic activity of the proteasome. Most rpn‐12(av93) hermaphrodites lack sperm leading to feminization of the germ line that can be partially rescued by mating to males. The lack of sperm phenotype can be suppressed by downregulation of TRA‐1, a player in the hermaphrodite germline sex determination pathway. Also, rpn‐12(av93) animals show significant nuclear accumulation of the meiotic kinase WEE‐1.3, a protein predominantly localized to the perinuclear region. Interestingly, chemical inhibition of the proteasome did not cause nuclear accumulation of WEE‐1.3. Conclusions RPN‐12 plays a previously unknown role in oogenesis and the germline sex determination pathway in C. elegans hermaphrodites.

Proteasomal subunit depletions differentially affect germline integrity in C. elegans

“…N-terminal GFP fusions with RPN-7, RPN-8, or RPN-9 showed ubiquitous expression in both the nuclei and cytoplasm of germline and somatic cells, including developing oocytes (Figure 7A and Supplemental Figure 6). This subcellular expression matches that determined by antibody staining against subunits of the proteasome core particle in C. elegans and in other systems (Brooks et al , 2000; Mikkonen, Haglund and Holmberg, 2017; Kumar and Subramaniam, 2018; Fernando, Elliot and Allen, 2020). Importantly, all three of these strains exhibited no effect on lifetime brood size and only a moderate reduction in lifespan when compared to wild-type control animals (data not shown).…”

“…With isoform RPN-6.1A being expressed only in the body wall muscles while RPN-6.1::OLLAS (which marks both Isoforms A and B) is expressed throughout the hermaphrodite female germ line but is distinctly absent from both sperm and the pharynx. Since downregulation of RPN-6.1 causes severe dysfunction of the proteolytic activity of the proteasome, we speculate that there is likely to be other RPN-6.1 variant(s) that functions in the pharynx and sperm (Vilchez, Morantte, et al , 2012; Fernando, Elliot and Allen, 2020). Indeed, RPN-6.2, a RPN-6 paralog, has recently been identified as sperm-specific (personal communication, Lynn Boyd).…”

“…Studies done in mammals and C. elegans show that the 19S RP lid subunit PSMD11/RPN-6.1 can regulate proteolytic activity of the proteasome modulating the production of the other proteasome subunits thus increasing or decreasing proteolytic activity of the proteasome (Vilchez, Boyer, et al , 2012; Vilchez, Morantte, et al , 2012; Lokireddy, Kukushkin and Goldberg, 2015). C. elegans studies have also uncovered proteasome subunits that are specific for germline development and fertility (Shimada et al , 2006; Pispa et al , 2008; Fernando, Elliot and Allen, 2020). RPN-10, RPN-12 and DSS-1 (RPN15/SEM1) were each shown to play specific roles in germline sex determination and oocyte development (Shimada et al , 2006; Pispa et al , 2008; Fernando, Elliot and Allen, 2020).…”

“…The oocytes briefly arrest at the diakinesis stage prior to maturation, ovulation, and completion of the meiotic divisions (Greenstein, 2005). Feeding L4 C. elegans hermaphrodites dsRNA against individual 19S RP proteasome subunits results in F1 progeny lethality for most of the 19S RP subunits, the exceptions being RPN-9, RPN-10, RPN-12, DSS-1, and RPT-6 (Takahashi et al , 2002; Shimada et al , 2006; Pispa et al , 2008; Fernando, Elliot and Allen, 2020). Despite the impact on embryonic viability, the effect of 19S RP subunit depletion on the reproductive capabilities of the RNAi-treated hermaphrodite mothers has not been examined.…”

Proteolytic activity of the proteasome is required for female insect reproduction

“…The physiological functions for individual Rpns have been determined in yeast, Drosophila and Caenorhabditis elegans [31,[35][36][37][38]. For instance, C. elegans RPN-12 is required for reproduction [35]. Yeast Rpn11 has metalloisopeptidase activity [31].…”