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.

Fig. 1.

A genetic assay that selects for the restoration of solubility to an insoluble protein. (A) A schematic depicts representative CAT fusion proteins. AID and AID-CAT are insoluble and, thus, produce chloramphenicol-sensitive cells; Cif and Cif-CAT are soluble allowing cells to be resistant to chloramphenicol; ADAT3 and ADAT3-CAT are insoluble unless coexpressed with ADAT2. (R, resistant; S, sensitive). (B) Plasmids carrying the indicated genes were transformed into BL21ai E. coli, plated on chloramphenicol containing plates under induction conditions. Resistant colonies were counted. Shown here are colony numbers obtained on LB plates containing 120 μg/mL chloramphenicol. (C) Chloramphenicol-resistant colonies after cotransformation of the indicated expression plasmids were counted. Coexpression of AID-CAT and the Mdm2 RING domain allowed for efficient survival on chloramphenicol plates. These interactions were revealed even when the Mdm2 RING plasmid was diluted 1:20 and 1:200 in a minilibrary of known noninteractors. For B and C, graphs represent the number of chloramphenicol resistant colonies per 1,000 cells plated.

Fig. 2.

B-cell cDNA library screen for factors that rescue the solubility of the AID-CAT fusion protein. (A) A schematic depicts the cloning and expression of a RAMOS cell-derived cDNA library along with AID-CAT. The full-length cDNA library was cloned into the attL sites of a modified pCDF-duet entry vector by using the Gateway system (Invitrogen). AID-CAT was cloned into a pCDF-duet modified destination vector. Recombination between these vectors is achieved by the addition of the LR Clonase Enzyme Mix, which uses the attR and attL recombination sites. After recombination, the final expression vector contains both a candidate from the cDNA library and AID-CAT. Bacteria are then transformed, selected in spectinomycin to assure that they contain the recombined vector and replica plated on Petri dishes containing IPTG/arabinose to induce expression and chloramphenicol to select for solubility. (B) Pathways representative of putative interactors, which were sequenced from the screen and independently confirmed in bacterial coexpression assays. (C) Cartoon of RNF126 domain structure. The position and sequences of the RING domain and Zinc finger domain are shown. C Inset shows that the amino acid composition of the C-terminal serine-rich domain is evolutionarily conserved.

Fig. 3.

RNF126 interacts with AID and its expression is induced in B cells after stimulation. (A) AID copurifies with RNF126 upon coexpression and purification in E. coli. Flag-tagged AID was coexpressed with His-tagged RNF126. Purification of RNF126 on a Talon cobalt column and elution with imidazole reveals that AID coelutes with RNF126. (B) RNF126 interacts with AID in a HEK 293T mammalian system. Flag-tagged AID and HA-tagged RNF126 were coexpressed. Affinity purification of either tag resulted in the purification of the binding partner only when both proteins were expressed. (C) Expression of RNF126 is induced in switching B cells after stimulation for class-switch recombination as assessed by qRT-PCR (normalized to CD19 levels). (D) RNF126 protein levels increase in murine B cells after stimulation to undergo class switch recombination in an AID-independent manner. Arrow denotes RNF126 band (Tubulin: loading control). The values beneath the blot denote a quantification of RNF126 protein levels, normalized to Tubulin.

Fig. 4.

RNF126 ubiquitylates AID. (A) AID is ubiquitylated by RNF126 in HEK 293T cells. Coexpression of hAID with HA-tagged RNF126 and Flag-tagged ubiquitin results in the formation of ubiquitylated AID, even in the presence of K48R/K63R mutant ubiquitin. RIPA extracts were prepared and AID immunoprecipitated with an anti-AID antibody. An anti-AID and an anti-FLAG (ubiquitin) blot are shown. The heavy chain band is marked as “HC.” (B) AID is ubiquitylated by RNF126 in vitro. Purified components of the ubiquitin reaction are incubated at 37 °C. (B Left) All components are necessary for ubiquitylation to occur (lane 1). The absence of individual components (lane 2–7) prevents ubiquitylation. (B Right) RNF126 monoubiquitylates AID. Addition of a mutant ubiquitin in which all lysines are mutated to arginine (K0 Ub) shows that the laddering is representative of multiple monoubiquitylation events. An anti-AID immunoblot is shown.

Fig. 5.

RNF126 selectively modifies AID compared with other B-cell associated E3 ligases. (A) The 293T cells were transfected with Flag-AID, alone or in combination with HA-RNF126 or HA-RNF8 (Left), and Flag-PCNA, alone or in combination with the same ligases (Right). Coexpression of RNF126, but not RNF8, results in AID ubiquitylation. Ubiquitylation of PCNA occurs upon coexpression of RNF8 (lane 3), but not RNF126 (lane 2). Both AID and PCNA were immunoprecipitated with αFlag and blotted with αAID and αFlag, respectively (Top). Western blots of AID (αAID), PCNA (αFlag), RNF126/RNF8 (αHA), and tubulin (αTubulin) of input (whole cell extract) are shown in the bottom three panels. Asterisks mark the unmodified and modified bands of AID and PCNA, and “HC” denotes the heavy chain band. Lane 1 of each image represents the expression of the substrate without exogenous ligase. (B) GST-RNF8 and GST-PCNA were used as an alternate ligase and substrate in the in vitro ubiquitylation assay. Reactions were carried out with either UbcH5b or UbcH5c as the E2 enzyme. In both cases, RNF8 selectively ubiquitylates PCNA and not AID (lanes 3 and 5) and RNF126 selectively ubiquitylates AID and not PCNA (lanes 4 and 6). Lane 1 excludes all components except the substrate and lane 2 only excludes the E3 ligase. Unmodified and modified bands are denoted with an asterisk. (C) BCA2, a homologous E3 ligase, cannot ubiquitylate AID. AID is expressed in 293T cells alone (lane 1) or in the presence of either RNF126 (lane 2) or BCA2 (lane 3). Only coexpression with RNF126, but not BCA2, results in ubiquitylation. The top image represents an αFlag IP and αAID immunoblot. Western blots of AID (αAID), RNF126/BCA2 (αHA), and tubulin (αTubulin) in the input/whole cell extract are shown in the bottom three images. The heavy chain band is marked with “HC.”