ubiquitination pathways. Mutation of mat-1, which encodes the CDC27 subunit of the APC, causes a diminution in the size of the fan and sensory rays similar to the defect produced by uba-1(it129) [29]. The heterochronic gene lin-41, which encodes a homolog of the RING finger subclass of E3 ligases, is also required for proper formation of the male tail. A decrease in LIN-41 function causes precocious retraction of the male tail so that the fan and rays are reduced or absent [65,66]. The DBL-1/TGF-ß pathway (men- tioned above) that determines body size also plays a role in formation of the spicules [67], and might be implicated in the protruding spicule phenotype of uba-1(it129) males. The late-onset paralysis and associated lethality produced by the uba-1(it129) mutation is unusual in two regards: it is sex-specific, affecting only males, and can be induced after all somatic development is complete. There are few reports of such post- developmental phenotypes for C. elegans, and this property suggests a defect in the maintenance of neuronal and/or muscle function rather than its establishment. Roles for ubiquitination in C. elegans neuromuscular activity have been reported previously. Multiple E2 conjugating enzymes have been implicated in polyglutamine protein aggregation in muscle [68]. E3 ligase complexes that have been demonstrated to affect either muscle or neuronal function include CHN-1/UDF-2, APC, KEL-8/CUL-3, SCF/FSN-1/ RPM-1, SCF/LIN-23, and SCF/SEL-10 [15,16,69–72]. Howev- er, the paralysis of uba-1(it129) males is distinct from the more subtle neuromuscular defects reported for other ubiquitin pathway components such as APC (decreased duration of forward movement) or KEL-8/CUL-3 (changes in nose touch response and spontaneous reversal frequency) [15,16]. Furthermore, functional roles for all of these enzymes have been demonstrated in hermaphrodites, so the sex-specific ubiquitination that is responsible for male paralysis remains to be elucidated. Why are male-specific processes, including the fertility defect of the male gamete (i.e., sperm), so acutely sensitive to the level of UBA-1 activity? One intriguing possibility involves the recently discovered role for ubiquitin-mediated proteolysis in the sex determination pathway. The TRA-1 transcription factor is the critical regulator of somatic and germ line sex determination and acts primarily as an inhibitor of male sexual fate [73]. Three FEM proteins negatively regulate TRA-1 activity and thereby promote male cell fates, including sperm development in hermaphrodites [74]. Starostina et al. [12] demonstrate that the FEM proteins form an E3 ubiquitin ligase complex with CUL-2 that binds to and promotes proteasome- dependent degradation of TRA-1. Impairment of UBA-1 function by mutation would be predicted to decrease activity of the FEM/CUL-2 E3 complex, leading to an increase in TRA-1 levels that would inhibit male developmental processes. This weakly feminizing effect might act synergistically with one or more of the E3 pathways described above. If this hypothesis is correct, then some of the sex-specific defects of the uba-1 mutation might be suppressed by a decrease in TRA-1 activity. The observation of synthetic embryonic lethality between uba- 1(it129) and mutations in components of the APC suggests a powerful approach for identifying new functions for downstream components of the ubiquitin pathway. A number of E2 and E3 homologs exhibit detectable phenotypes in genome-scale RNAi screens, but the majority are indistinguishable from wild type [34,35]. One possible explanation is that many of these enzymes are functionally redundant, and that the determination of their roles will require inactivation of multiple E2s or E3s. Alternatively, in some instances the reduction of E2 or E3 levels by RNAi might be insufficient to disrupt function. However, the uba-1 mutation provides a sensitized genetic background for detecting decreased activity of downstream enzymes. Reanalysis by RNAi screening of UBA-1 in C. elegans Development the E2 and E3 homologs in the uba-1 mutant strain is likely to reveal novel functions for a number of those genes whose roles are currently unknown. Materials and Methods Genetics C. elegans strains were derived from the wild-type isolate N2 (Bristol) and contained one or more of the following mutations: uba-1(it129)IV, uba-1(ok1374)IV, dpy-20(e1282)IV, fem-1(hc17)IV, fem-3(q20)IV, him- 5(e1490)V, mat-3(or180)III, fzy-1(h1983)II, spe-26(it112), or chromo- some IV deficiencies eDf19 or mDf7. A linked uba-1(it129) dpy- 20(e1282) double mutant strain was generated to facilitate discrim- ination of homozygous and heterozygous lines in some experiments. The integrated oma-1::GFP transgenic line was constructed by Reuyling Lin [54]. Strains were maintained on NGM plates seeded with E. coli strain OP50. Age-synchronized populations of embryos were obtained by sodium hypochlorite treatment of gravid hermaphrodites. Strains were maintained at 15uC and shifted to 25uC as indicated for phenotypic analysis. Genetic manipulations were carried out according to Brenner [75]. Microscopy Microscopy was performed with an Olympus BX51TF or Zeiss Axio Imager equipped with Nomarski DIC objectives and appropriate filter sets for fluorescent imaging and cooled CCD camera for image capture. Images were processed using the AxioVision (release 4.6) package and prepared for publication using Adobe Photoshop CS v. 9.0.2. Intact animals were typically mounted on 2% agarose pads for imaging. Body length was measured from captured images using ImageJ software v. 1.38. Sperm Assays Sperm morphology was assessed by dissection of gonads from adult hermaphrodites or males in SM medium [43]. Nuclear DNA morphology was visualized by DAPI staining of sperm from dissected gonads. In vitro activation of male spermatids was by treatment with monensin on poly-lysine-coated slides [43]. Motility and localization of hermaphrodite sperm were deter- mined in intact animals by fixation and staining with DAPI, then counting the number of sperm nuclei in the spermathecae and uterus. Sperm transfer was ascertained by vital staining of males [76] with the mitochondrial dye MitoTracker Red CMXRos (Molecular Probes), then mating to unstained hermaphrodites anesthetized with tricaine and tetramisole. After 12 or 24 h, fluorescently-labeled male sperm within the hermaphrodite reproductive tract were visualized by microscopy using rhodamine filters. Self-fertility of hermaphrodites was assessed by shifting individual L3 animals to 25uC and counting the entire brood size. Cross-fertility of males was determined by mating with individual wild-type hermaphrodites or fem-1(hc17) females, then counting the number of male and hermaphrodite progeny produced by each animal after mating. Cloning and Molecular Analysis The uba-1(it129) mutation was localized to chromosome IV between elt-1 and dpy-20 by three-factor crosses. Single nucleotide polymorphisms that affect restriction sites (snip-SNPs) were employed as physical mapping markers of individual uba-1(it129) dpy-20(e1282) recombinants with Hawaiian strain CB4856 [44]. Deficiency mapping was performed by complementation testing in uba-1(it129)/Df heterozygous strains. RNAi of candidate genes was performed by feeding [77] and assessed by phenocopy of F1 embryonic lethality for treated adult hermaphrodites and by PLoS Genetics | www.plosgenetics.org 11 July 2008 | Volume 4 | Issue 7 | e1000131