C. elegans hermaphrodites are essentially XX females that evolved the ability to briefly produce sperm in the same germ line that produces oocytes [1]. XX spermatogenesis requires a specific protein/mRNA ternary complex composed of an RNA binding protein, GLD-1, an F-box protein, FOG-2, and the mRNA product of the feminizing gene
tra-2. GLD-1 and FOG-2 bind the 3' UTR of
tra-2 mRNA and repress its activity during spermatogenesis [2,3,4]. Loss of GLD-1, FOG-2, or the GLD-1-binding sites in the
tra-2 3' UTR eliminates sperm production. GLD-1 is a highly conserved regulator of meiotic progression and oogenesis, and therefore has many RNA targets [5]. In contrast, FOG-2 is a recently evolved C. elegans-specific F-box protein [2, 6] only necessary for hermaphrodite spermatogenesis. It has no known role in males, although they express it [7]. The precise role of FOG-2 in regulating germline sex is unknown, but there are clues. FOG-2 interacts with SKR-1, a component of the E3 ubiquitin ligase complex [6]. This suggests it acts as a canonical F-box protein by targeting another protein for ubiquitin-mediated degradation. However, their similar feminized phenotypes indicated GLD-1 is not that target. If FOG-2 is a regulator of proteolysis, then its F-box should be crucial to its function. We deleted 57 nucleotides encoding 19 conserved amino acids of the FOG-2 F-box via CRISPR. This strain was indeed Fog, but subsequent immunoblotting determined the deleted protein was unstable. Future work will target individual conserved residues with missense point mutations. As another approach to elucidating FOG-2's mechanism of action in sex determination, we are currently seeking its putative ubiquitination target(s) by identifying protein interactors. We are employing both targeted yeast two- hybrid assays and unbiased immunoprecipitation and protein mass spectrometry using a 3XFLAG::TEV::6HIS tagged strain of FOG-2. Recent work from the Haag Lab suggests that FOG-2 may have roles independent of its association with the
tra-2 3' UTR, and potentially independent of GLD-1 as well [8]. This led us to wonder what fraction of FOG-2 interacts with GLD-1 and
tra-2 mRNA. We stained the germ line using sm-FISH to
tra-2 mRNA and antibodies to HA-tagged FOG-2 and to GLD-1. Surprisingly, FOG-2, GLD- 1 and
tra-2 do not obviously co-localize. These experiments are consistent with a mode of FOG-2 action other than the aforementioned ternary complex, but much remains to be learned. 1. Ellis, R.E. and S.Y. Lin, F1000Prime Rep, 2014. 6: p. 62. 2. Clifford, R., et al., Development, 2000. 127: p. 5265-5276. 3. Jan, E., et al., EMBO J., 1999. 18(1): p. 258-69. 4. Lee, M.H. and T. Schedl, Genes Dev, 2001. 15(18): p. 2408-20. 5. Francis, R., et al., Genetics, 1995. 139(2): p. 579-606. 6. Nayak, S., etal., PLoS Biology, 2005. 3: p.
e6. 7. Schedl, T. and J. Kimble, Genetics, 1988. 119: p. 43-61. 8. Hu, S., et al., Developmental Biology, 2018.