Supplemental Material

This article contains the following supporting material:

• Figure S1 - The effects of continuous knockdown of the rpn-1, an essential subunit gene of the 26S proteasome. Among any extent of partial inhibition of rpn-1, no individuals showed specific defects in fertilization as those seen in rpn-10 (RNAi). At 5% feeding of bacteria expressing double strand RNA of rpn-1, no signs of abnormality in reproductive system were observed and viable embryos were produced. At 10% feeding, many lethal F1 embryos were produced without retardation of fertilization. At 20% or higher feeding, P0 worms exhibited highly sick morphology and no reproductive systems were observed in these worms.
• Figure S2 - rpn-10 (tm1180), a mutant worm expressing a C-terminally truncated form of RPN-10, shows a sperm-defective phenotype. (A) Schematic diagram of RPN-10 proteins encoded by wild-type (WT) and tm1180 mutant. Mutant RPN-10 (ΔC RPN-10) contains an N-terminal VWA domain but lacks the C-terminal domain including polyubiquitin (poly-Ub)-binding UIMs. (B) rpn-10 (tm1180) mutant worms express a truncated form of RPN-10 (left panel, arrow) but accumulate polyubiquitinated (poly-Ub) proteins (right panel). (C) The average numbers of progeny per parent at 25°C are indicated. The number from the N2 wild-type worm is defined as 100%, and relative numbers are presented. Note that ufd-2 (RNAi) results in a nearly complete sterile phenotype in rpn-10 (tm1180) mutant but did not affect the progeny from N2 wild-type worms. (D) rpn-10 (tm1180) mutant young adult worms show expanded oocytes (indicated by a red box) and a nearly empty uterus (indicated by a yellow box). (E) Hoechst staining of dissected gonads shows reduced number of sperm in rpn-10 (tm1180). Spermatheca is indicated by a white box. Arrow indicates the absence of sperm at the position of spermatheca. Scale bar, 10 μm.
• Supplemental Table 1 - The effects of continuous knockdown of the rpn-1. Among any extent of partial inhibition of rpn-1, no feminization defects from healthy worms were observed, while knockdown of rpn-10 induce sterility without affecting viability of the parental worms. The results of % removal of transcripts were evaluated from the quantification results of RT-PCR against corresponding genes.
• Supplemental Table 2 - The effects of continuous knockdown of the rpn-1. Among any extent of partial inhibition of rpn-1, no synthetic feminization was observed with ufd-2, while clear synergism was observed in the co-knockdown of rpn-10 and ufd-2 genes.