Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

Abstract

Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.ubiquitin | immunology | lymphocyte M ost large-scale protein-protein interaction data published to date has been produced with yeast two-hybrid assays (1-3) and in vivo pull-down approaches followed by mass spectrometry to identify proteins that coprecipitate with the protein of interest. Although these purification methods were first developed for small-scale protein identification experiments, they have been successfully adapted for use in genome-wide proteomics studies (4-6). Proteins that are poorly soluble or insoluble when ectopically expressed are least amenable to characterization using these tools. Improper folding or exposure of hydrophobic domains can result in insolubility when proteins are expressed individually; however, soluble complexes can form upon coexpression of a native partner. Based on this idea, we have devised a high-throughput protein-protein interaction screen, which involves the coexpression and cofolding of two proteins: an unknown protein expressed from a cDNA library and a known, insoluble "bait" fused to a protein that confers antibiotic resistance. Thus, solubility is directly linked to a traceable marker. We apply this technique to identify cofactors for activation-induced cytidine deaminase (AID), an enzyme that deaminates deoxycytidines in single-stranded DNA.AID initiates both somatic hypermutation (SHM) and classswitch recombination (CSR) during antibody diversification (7,8). A number of cofactors have been identified by a combination of genetic and proteomic approaches, which suggest that AID synchronizes with other broadly important cellular pathways including, but not limited to, RNA splicing and processing (9, 10), cellular trafficking (11)(12)(13)(14), and transcription/RNA polymerase stalling (8,(15)(16)(17); however, a concise picture of the players involved in the AID reaction and the...

E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells

“…3C). A fraction of AID is ubiquitinated in B cells, as has been reported previously by other groups (Aoufouchi et al 2008;Delker et al 2013). These results demonstrate that in CSR-stimulated B cells, a portion of cellular RNA polII is ubiquitinated by Nedd4.…”

AID downregulation is a novel function of the DNMT inhibitor 5-aza-deoxycytidine

“…Whether APC/C Cdh1 E3 ligase is also involved in the AID polyubiquitination and degradation induced by 5-aza-CdR requires investigation. Recently, the E3 ligase RING finger protein 126 (RFN126) has been found for the ubiquitination of AID [38]. Whether 5-aza-CdR could upregulate RFN126 to degrade AID will also be investigated.…”

E3 Ubiquitin ligase RNF126 regulates the progression of tongue cancer

“…RNF126 is an RING type E3 ligase which contains a Zn finger near the N‐terminus and a RING finger at the C‐terminus 8. It expresses in both the nucleus and the cytoplasm and is involved in a diverse set of cellular processes.…”