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Characterisation of Caenorhabditis elegans RING Finger Protein 1 (RFP-1), a binding partner of ubiquitin-conjugating enzyme 1 (UBC-1) Crowe, Emily

Abstract

Ubiquitin-mediated proteolysis is an ATP-dependent pathway that targets proteins for degradation in eukaryotes. The conjugation of a multiubiquitin chain to a target protein requires a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and often a ubiquitinprotein ligase (E3). While specificity for target protein recognition resides in the E2 and E3 components, very little is known about their roles in multicellular organisms. In a yeast two-hybrid screen, RING Finger Protein 1 (RFP-1), UBR1, and SCA-1 were identified as potential binding partners of C. elegans UBC-1, a ubiquitin-conjugating enzyme with a high degree of identity to S. cerevisiae UBC2/RAD6. The interaction between RFP-1 and UBC-1 was confirmed by co-immunoprecipitation experiments in buffer and C. elegans extract. The RFP-1 and UBC-1 interaction was not dependent on the presence of ubiquitin or ATP. Yeast interaction trap experiments mapped the region of interaction to the basic N-terminal 313 residues of RFP-1 and the UBC core domain of UBC-1. Western blot analysis and indirect immunohistochemical staining showed that RFP-1 is present in embryos, larvae, and adults, where it is found in intestinal, nerve ring, pharyngeal, gonadal, and oocyte cell nuclei. Limited UBC-1 indirect immunohistochemical staining experiments showed that UBC-1 is localised to oocyte nuclei. Double stranded RNA interference experiments against rfp-1 indicate that this gene is not essential, but is required for proper vulval development and for egg-laying. By contrast, RNA interference experiments against ubc-1 gave no obvious phenotype, suggesting that ubc-1 is nonessential or is functionally redundant. The Class A and Class B Synthetic Multivulva pathways define two functionally redundant genetic pathways in C. elegans that antagonise vulval development. RNA interference experiments against rfp-1 in lin-15A (n767) or lin-15B (n744) animals suggest that rfp-1 is neither a Class B nor a Class A Synthetic Multivulva gene. Similarly, RFP-1 failed to directly interact with LIN-35 (467-961), a Class B Synthetic Multivulva protein and the putative retinoblastoma protein in C. elegans. The rfp-1 RNA interference-induced phenotypes were exacerbated in the lin-15B (n744) background. Based on the sequence similarity to R. norvegicus Staring and S. cerevisiae Brelp, RFP-1 may be a RING finger ubiquitin-protein ligase required for UBC-1-dependent ubiquitylation of nuclear proteins.

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