Ubiquitin specific proteases (USPs) of family C19 is important for the regulation of the processes in which ubiquitin (Ub) is involved. C. elegans has 26 genes that code for the proteins of this family. We performed RNAi on 21 of them. The most severe effect was observed by RNAi of F09D1.1 (100% embryonic lethal). The gene product of F09D1.1, however, is predicted to be U4/U6.U5 tri-snRNP-associated protein with no protease activity because the catalytic residue is not conserved. Some mildly abnormal phenotypes were observed by RNAi of several other genes. Feeding RNAi of
duo-1 caused no abnormality in the P0 and F1 animals but 80% lethality on the F2 embryo. The knockout mutant
duo-1 (
tm934 ) showed 80% larval lethality. Thus,
duo-1 is thought to be important for viability of C. elegans . The
duo-1 gene codes for a protein of 822 amino acid residues, which is predicted to have a unique domain structure; it has an insertion of an OTU-like cysteine protease domain in the USP domain. Since OTU-like cysteine proteases have been reported to be a novel type of deubiquitinating enzyme, DUO-1 is presumed to have dual catalytic sites for ubiquitinated proteins. To confirm this, the OTU-like cysteine protease domain was expressed as a GST-fusion protein in E. coli and purified. The recombinant OTU domain showed C-terminal hydrolase activity toward recombinant Ub fused with HSV and His-tags at the C-terminus, but not toward SUMO-1- and NEDD8-fused substrates. To evaluate the ability of each catalytic site to cleave branched poly-Ub chains, we performed in vivo isopeptidase assay by co-expression of HA-tagged Ub and DUO-1 in the COS7 cells. Upon expression of wild-type DUO-1, Ub-conjugate level in the transfected COS7 cytosol was found to decrease. When Cys223 and Cys607, the predicted catalytic residues in the USP and OTU domains, respectively, were individually replaced with Ala, a low level of Ub-conjugates was observed on each case, while the double mutation of C223A and C607A showed a high level of Ub-conjugates. These results suggest that each of the predicted catalytic sites of DUO-1 has isopeptidase activity toward polyubiquitylated proteins.