Functional requirements of AID’s higher order PNAS PLUS structures and their interaction with RNA-binding

Samiran Mondala, Nasim A. Beguma, Wenjun Hua, and Tasuku Honjoa,1

aDepartment of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan

Contributed by Tasuku Honjo, February 3, 2016 (sent for review October 27, 2015; reviewed by Atsushi Miyawaki and Kazuko Nishikura)

Activation-induced (AID) is essential for the interaction, enabling AID to exert distinct physiological functions somatic hypermutation (SHM) and class-switch recombination (CSR) through its association with cofactors. Regrettably, however, there of Ig . Although both the N and C termini of AID have unique is little structural information available that can explain any of functions in DNA cleavage and recombination, respectively, during AID’s regulatory modes of action, including its association SHM and CSR, their molecular mechanisms are poorly understood. mechanisms, in the context of its physiological functions. Using a bimolecular fluorescence complementation (BiFC) assay Although a significant amount of structural information is available combined with glycerol gradient fractionation, we revealed that for a number of APOBEC family members, the 3D structures of A1 the AID C terminus is required for a stable dimer formation. Further- and AID are yet to be resolved (19, 20). The CDD family of more, AID monomers and dimers form complexes with distinct exists in nature in a variety of structural forms, including monomeric, dimeric, and tetrameric forms, and comparative structural modeling heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers using the yeast CDD structure predicts a dimeric structure for both associate with DNA cleavage cofactor hnRNP K whereas AID dimers A1 and AID (21, 22). On the other hand, homology modeling with associate with recombination cofactors hnRNP L, hnRNP U, and the APOBEC2 (A2) crystal structure, which seems to be a tetramer Serpine mRNA-binding 1. All of these AID/ribonucleoprotein composed of two head-to-head interacting dimers, predicts that AID ’ associations are RNA-dependent. We propose that AID s structure- forms a tetramer (23). Notably, A2waslaterreportedtoexistasa specific cofactor complex formations differentially contribute to its monomer in solution (24). Similarly, an atomic force microscopic DNA-cleavage and recombination functions. (AFM) study found that AID exists in the cell predominantly as a monomer associated with a single-strand DNA (25). AID | BiFC | hnRNP U | SERBP1 | APOBEC However, the same AFM dataset was interpreted differently by an- other group of investigators, who concluded that AID probably forms ’ ctivation-induced cytidine deaminase (AID), which is expressed an A2-like tetramer in solution (26). The modeling of AID scatalytic pocket in reference to eight APOBEC family members suggested Ain antigen-stimulated mature B cells, is essential for Ig somatic – hypermutation (SHM) and class-switch recombination (CSR) (1, 2). that most of the AID DNA complex remains in an inactive state due AID induces DNA breaks at the variable (V) and switch (S) regions to occlusion by the substrate DNA, which may explain its weak cat- alytic activity for cleaving DNA in vitro (27). during SHM and CSR, respectively (3, 4). Although both processes One of the limitations of the computational modeling of AID’s are initiated by AID-induced DNA cleavage, point at the structure is that AID’s N-and C-terminal sequences are sub- V region are executed mostly by error-prone DNA repair whereas stantially different from those of other APOBEC members and CSR is accomplished by recombination of cleaved ends at donor thus reside outside the modeling template. Although the struc- and acceptor S regions (5, 6). However, the detailed mechanisms tural outcome of a protein can differ by a variety of reasons, by which AID carries out the two mechanistically distinct functions including the methods applied (28), none of the AID studies for SHM and CSR have yet to be uncovered (7). Studies on AID mentioned above explain why the C-terminal deletion of AID ’ INFLAMMATION mutants revealed that AID s N- and C-terminal domains are dis- leads to the loss of CSR function only. Therefore, model-based IMMUNOLOGY AND tinctly required for its DNA-cleavage and recombination functions, computational simulation may not explain the physiological respectively (8–10). Mutations at the N terminus of AID impair structure–function relationship of AID in B cells. SHM as well as CSR whereas those at the C terminus abrogate Here, we explored AID’s structure–function relationship using CSR only and show increased SHM activity. Recent studies dem- a bimolecular fluorescence complementation (BiFC) assay, onstrated that the CSR process after DNA cleavage, including the synapsis formation between cleaved ends, is impaired with the Significance C-terminally defective AID, indicating that AID’s C terminus confers a CSR-specific recombination function, independent of ’ This paper demonstrates that activation-induced cytidine de- AID s DNA cleavage function, by an unknown mechanism (11, 12). aminase (AID), an essential in antigen-induced antibody AID belongs to the APOBEC ( mRNA-editing diversification, forms distinct ribonucleoprotein complexes de- enzyme catalytic polypeptide) family of cytidine deaminases pending on its structural states: namely monomers or dimers. The (CDDs) and shows high with APOBEC1 (A1) identified RNA-binding proteins are required for the function of (1, 13, 14), which edits apolipoprotein B (APOB) mRNA. The AID: namely DNA cleavage or recombination. In addition, the APOB mRNA editing ability of A1 is highly dependent on its complex formation between AID and heterogeneous nuclear ri- cofactors, A1CF/ACF (15, 16) and RBM47 (17), both of which bonucleoproteins (hnRNPs) is RNA-dependent. belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Recently, two A1CF-like hnRNPs, hnRNP K and hnRNP Author contributions: S.M., N.A.B., and T.H. designed research; S.M., N.A.B., and W.H. L, were identified as the cofactors of AID and found to be in- performed research; N.A.B. contributed new reagents/analytic tools; S.M. analyzed data; volved in the cleavage and recombination of DNA, respectively and S.M., N.A.B., and T.H. wrote the paper. (18). Because the N and C termini of AID differentially regulate Reviewers: A.M., Brain Science Institute of RIKEN; and K.N., The Wistar Institute. two functions of AID—cleavage and recombination, respectively— The authors declare no conflict of interest. we speculated that the AID termini would be critical for function- 1To whom correspondence should be addressed. Email: [email protected]. coupled cofactor association. For instance, the N or C terminus of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. AID may function as a molecular switch that induces an AID–AID 1073/pnas.1601678113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1601678113 PNAS | Published online February 29, 2016 | E1545–E1554 Downloaded by guest on September 23, 2021 which detects homo- or heteromeric protein–protein interactions expression amount of all of the four constructs (Fig. 1D). We in live cells (29, 30). For the homomeric interaction assay, the observed that none of the fusion constructs alone produced a target protein is fused to two nonfluorescent halves of a green or fluorescence signal above background. We also verified that the red fluorescent protein. An interaction between two of the target coexpression of mKG.N and mKG.C did not produce any BiFC proteins brings the two nonfluorescent halves of the fluorescent signal (Fig. S1D). Furthermore, the efficiency of the AID–AID protein into close proximity, reconstituting the fluorescence. The interaction was assessed by a direct co-IP analysis (Fig. 1E), which BiFC assay thus allows a rapid analysis of the dimerization of a mostly agreed with the BiFC MFI profile (Fig. 1 B and C). protein of interest in live cells. Fluorescence microscopic observation showed that the BiFC sig- By combining this assay with other biochemical approaches, nal of AID was localized to the cytoplasm (Fig. S1C), which was such as coimmunoprecipitation (co-IP) and glycerol gradient previously shown to be the major localization site of AID (Fig. sedimentation, we revealed the presence of both monomeric and S1C) (41). The expected subcellular localizations were observed dimeric forms of AID in analyzed cells. Intriguingly, C-terminal for the other APOBECs, confirming that the BiFC signals rep- AID mutants that lost CSR function showed a severe dimerization resent native characteristics of the APOBEC proteins. defect, suggesting that AID’s C terminus is required to stabilize the dimeric structure that is required for CSR. We also showed AID C-Terminal Mutants That Are Defective in CSR Are Unable to Form a that the AID monomer and dimer associate with different RNA- Dimer. To evaluate the functional relevance of AID dimer for- binding proteins (RBPs) to form ribonucleoprotein (RNP) com- mation, we examined the BiFC signals using AID mutants that plexes. Based on these findings, we propose that the monomeric showed clear functional defects. AID C-terminal mutants are of AID–RNP complex includes hnRNP K (18) and contributes to the particular importance because they lose CSR function but retain DNA cleavage function of AID whereas the dimeric AID–RNP strong SHM activity (Fig. 2A). Such well-characterized C-terminal complexes include hnRNP L (18), hnRNP U (31), or Serpine AID mutants include JP8Bdel (R183X), P20 (34-aa insertion at mRNA-binding protein 1 (SERBP1) (32) and contribute to the residue 182), JP41 (R190X), and JP8B (26-aa frameshift re- recombination step of CSR. placement at residue 183) (8). Remarkably, each mutant showed severely defective BiFC signal generation in all four pairwise Results combinations even though they were all expressed well (Fig. 2 B and Detection of APOBEC Homodimer Formation by the Monomeric Kusabira C). These results clearly demonstrated that the C-terminal muta- Green-BiFC Assay. The monomeric Kusabira Green-BiFC (mKG- tions severely impaired the homodimer-formation ability of AID in BiFC) assay detects the association of two nonfluorescent mKG live cells. In addition, all of the C-terminal mutants were also de- fragments (mKG-N and mKG-C), which occurs through the in- fective in their heteromeric interaction with WT AID (Fig. 2E). To teraction of fusion partners (33). To validate the mKG-BiFC– confirm the loss of the homo- and heterodimerization abilities, we based protein–protein interaction system for the APOBEC family, performed co-IP analyses with the C-terminal mutants (Fig. 2 D and we first tested the four members of this family that are known F). Although the WT AID could efficiently pull down its WT AID to undergo homomeric interactions. The tetrameric structure of counterpart, none of the C-terminal mutants could pull down either APOBEC2 (A2), composed of two dimeric units, was revealed their respective counterparts or the WT AID. Thus, both the BiFC from its crystallographic structure (23). Biochemical studies and and co-IP analyses confirmed that the homo- and heterodimeriza- atomic force microscopy observations showed that APOBEC3G tion abilities of AID were lost in the C-terminal mutants. (A3G) formed a dimer (34, 35) or an oligomer (36). APOBEC3A (A3A) was very recently crystalized as a dimer (37). Also, the di- The AID N Terminus Is also Required for Dimer Formation. Although meric structure of APOBEC1 (A1) was suggested by biochemical the BiFC signal intensity did not necessarily reflect the proximity studies (13, 38, 39). between the two fusion termini of AID, the maximum signal We examined the dimer formation of A2, A3G, A3A, and A1 obtained by the combination of C (AID-mKG.N) and B (mKG.C- using the mKG-BiFC assay. We generated four BiFC constructs AID) might suggest the involvement of AID’s N terminus in the (A, B, C, and D) for each APOBEC family member, as depicted AID–AID dimer formation. Thus, we generated a CB pair of in Fig. S1A. The split fragments of mKG, designated mKG.N mKG-AID constructs, with serial deletions at the N terminus of (N-terminal half) and mKG.C (C-terminal half), were fused to the AID (ΔN5, ΔN10, and ΔN26), and examined their homodimer N or C terminus of the full-length APOBEC construct: A, mKG.N- formation by the BiFC assay (Fig. 3 A and B). The serial N-ter- APOBEC; B, mKG.C-APOBEC; C, APOBEC-mKG.N; and D, minal truncations caused a progressive loss of the BiFC signal APOBEC-mKG.C (Fig. S1A). As expected, pairwise (AB, AD, (Fig. 3 B and C) although all of the mutants were expressed well. CB, or CD) transfection of the fusion constructs in human em- Similar to the C-terminal mutants, the N-terminally truncated bryonic kidney (HEK) 293T cells generated BiFC-positive cells for AID mutants (ΔN5, ΔN10, and ΔN26) barely formed hetero- all of the ABOBECs (Fig. S1B). All of the A2–A2 interaction dimers with the WT AID (Fig. 3E). Co-IP analysis using homo- combinations produced BiFC with a high mean fluorescence in- meric and heteromeric combinations confirmed that the deletion tensity (MFI), which may reflect A2’s high-affinity homomeric of as few as 10 aa at the AID N terminus was sufficient to com- (di/tera) interaction property in live cells. The A3G–A3G in- pletely disrupt the homo- and heterodimeric interactions (Fig. 3 F teraction also produced intense BiFC signals, in agreement with a and G). Examination of the CSR efficiency of the N-terminal − − previous A3G-interaction study using an mCherry-BiFC assay mutants in AID / spleen B cells revealed a similar progressive (40). A3A and A1 showed relatively weaker but substantial BiFC loss of this activity (Fig. 3D). Taken together, these findings show signals. Interestingly, the maximum BiFC signal was obtained for that AID’s intact N terminus is important for the AID–AID in- A1 by the AB combination whereas the CD combination was the teraction and for CSR activity. most efficient for the rest. For each APOBEC, none of the indi- vidual mKG fusion constructs (A, B, C, or D) alone produced an Separation of AID Monomer and Dimer on a Glycerol Gradient. Although appreciable BiFC signal, confirming that the mKG-BiFC assay the BiFC signal in live cells indicated a homomeric interaction of specifically detected APOBEC–APOBEC dimerization. AID, it could not provide further information about the AID tertiary structure that might incorporate other proteins and RNA. Thus, we Evidence for AID Dimer Formation by the mKG-BiFC Assay. Using the performed glycerol gradient centrifugation of the cell extracts from mKG vector system, we next examined the AID–AID interaction in HEK293T cells expressing the CB combination of mKG-AID con- HEK293T cells. BiFC signals generated upon pairwise cotransfec- structs. In reference to the background fluorescence signal from tion of the AID–mKG fusion constructs were measured by HEK293T cell extracts, the CB combination of mKG-AID revealed FACS (Fig. 1 A and B). Remarkably, the CB pair produced the two major fluorescence peaks at around fractions 3–12 and 19–23, highest MFI (Fig. 1 B and C) although all of the four interacting which we called the low molecular weight (LMW) and high mo- combinations generated BiFC-positive cells with nearly the same lecular weight (HMW) regions, respectively (Fig. 4A).

E1546 | www.pnas.org/cgi/doi/10.1073/pnas.1601678113 Mondal et al. Downloaded by guest on September 23, 2021 Western blot analyses of the gradient fractions showed that PNAS PLUS the distribution of AID, fused with mKG-N and mKG-C frag- ments, in general corresponded well with the two fluorescence peaks (Fig. 4B), indicating that the reconstituted BiFC signal was stable after cell lysis. To distinguish the distribution of AID monomer and dimer, we performed IP using gradient fractions corresponding to the two major BiFC peak areas (Fig. 4C). Be- cause the two mKG-AID constructs were tagged with different epitopes (FLAG and HA), pulling down HA-fused AID by anti- FLAG IP, and vice versa, provided further evidence of dimerized AID in the individual fractions. The failure of reciprocal AID co- IP suggested the exclusive presence of the monomer. These results showed that the monomeric AID was exclusively distributed in fractions 3–4 whereas the dimeric form was predominantly in fractions 5–12 and fractions 19–23. In the gradient analyses of AID mutants defective at either the C terminus (P20 and JP8B) or N terminus (ΔN10), the BiFC signal was dramatically decreased at both the LMW and HMW regions compared with the WT AID (Fig. 4A), in agreement with the requirement of AID’s N and C termini for dimer formation (Fig. 4B), and the presence of dimer at the HMW region. The anti-FLAG IP of the P20 and JP8B mutants could not pull down their untagged counterparts (Fig. 4D), indicating that they did not form a homodimer, although they were broadly distributed between fractions 3 and 12. Because the AID C-terminal mu- tants were all shown to be monomeric, their wide distribution along the gradient suggested that some of the monomers asso- ciated with other proteins and . Collectively, these results showed that AID distributed in the LMW region could be sub- divided into a narrow region of exclusive monomer (fractions 3–4) and a broader region of monomer and dimer (fractions 5–12) and that the HMW fractions of AID seemed to contain dimers.

AID Forms Multiple RNP Complexes. To examine the multimeric complexes containing AID, extracts of HEK293T cells expressing the CB combination of mKG-AID constructs were first run on the glycerol gradient after RNase or high salt (500 mM) treatments. Strikingly, either RNase or salt treatment caused the BiFC peak in the HMW region to disappear completely, suggesting that the HMW region contains higher order AID–RNP complexes (Fig. 5A). In contrast, these treatments did not abolish the BiFC peak in the LMW region. Although the peak was slightly broadened, it was possibly due to contamination of AID species dissociated from the HMW complex. We next examined the distribution profiles of AID-interacting INFLAMMATION

RBPs (hnRNP I/PTBP1, SERBP1, PABP, hnRNP U, and hnRNP C) IMMUNOLOGY AND that we discovered during AID co-IP (18, 42) in addition to hnRNP K and hnRNP L, which are known to interact with AID through RNA. We generated the migration profiles of the RBPs under three different conditions: with or without AID and after RNase treatment (Fig. 5B). HnRNP K, hnRNP I, and SERBP1, but not hnRNP L, PABP, hnRNP U, or hnRNP C, showed a clear change in their distribution patterns in the presence of AID. The dis- tribution profiles of all of the RBPs were strongly affected by RNase treatment. Particularly, hnRNP U and hnRNP C, which were normally found in the HMW region, shifted dramatically to Fig. 1. Evidence of AID dimer formation in living cells by the mKG-BiFC assay. the LMW region. (A) Schematic of the AID–AID dimerization examined by the mKG-BiFC The direct co-IP analysis of whole-cell extracts confirmed the assay. Fluorescence reconstitution (green) occurs when the nonfluorescent N- and association of these RBPs with AID, and their interactions were C-terminal mKG fragments (mKG.N and mKG.C) are assembled by AID homo- highly sensitive to both RNase and salt treatment (Fig. 5C). dimerization. The two mKG fragments fused to full-length AID (light orange) are Notably, these treatments did not affect AID dimer formation, shown in cyan and yellow, respectively. The diagram below shows the four types – given that the Flag IP of tagged AID (B construct) could pull (types A D) of fusion constructs used in the mKG-BiFC assay for AID. In these down its untagged counterpart (C construct), suggesting that the constructs, AID was placed either at the C or N terminus of the mKG fragments, and the constructs were transfected into HEK293T cells alone or in combination as BiFC-mediated AID dimer was quite stable once formed. All of indicated above each FACS profile (B). Values inside the histogram plots represent the RBPs examined were almost completely dissociated from AID after RNase or salt treatment, except hnRNP M, which the mean fluorescence intensity (MFI) of the BiFC-positive cells. (C) Mean fluo- A rescence data of the BiFC-positive cell population from three independent ex- formed a complex with AID at the HMW region (Fig. S2 and periments are shown as bar graphs. (D) Western blot analysis of the mKG–AID B). No association of AID with hnRNP Q or hnRNP E1/PCBP1 fusion constructs. (E) AID dimer formation detection by the anti-FLAG IP of A and was detected (Fig. S2 A and B). C coexpressed with B and D, which had a FLAG epitope. Coprecipitated proteins We also tested whether the monomeric C-terminal mutants were detected by immunoblotting with an anti-AID antibody. could interact with the RBPs, by performing co-IP experiments of

Mondal et al. PNAS | Published online February 29, 2016 | E1547 Downloaded by guest on September 23, 2021 Fig. 2. AID C terminus mutants are defective in di- merization. (A) Structures of AID C-terminal mutants and their CSR and SHM efficiencies in reference to WT. The CSR and SHM efficiencies of the mutants are from published reports. (B) FACS analysis of HEK293T cells transfected with pairwise combinations of the indicated mKG fusion constructs of WT AID or C-terminal AID mutants. MFI values of BiFC are shown in the respective histogram plots. The ex- pression level of each mutant is shown next to its FACS profile. Tubulin was used as a loading control. (C) Percentage of MFI (mean ± SD; n = 3) is shown only for the CB combination. (D) Immunoprecipita- tion to analyze the homodimerization of the AID C-terminal mutants was performed by coexpressing the CB combination of BiFC constructs in HEK293T cells. The FLAG epitope was fused to the C terminus of the AID mutants in the B constructs. Cell lysates were IPed with anti-FLAG M2 agarose, and the coprecipitated proteins were analyzed by immuno- blotting with an anti-AID antibody. (E) BiFC assay to determine the heterodimer formation between WT AID and the C-terminal mutants. Frequency his- tograms of HEK293T cells cotransfected with the CB combination of BiFC constructs, as indicated above each plot. The data from three independent experiments are shown next to the FACS profile. (F) Immunoprecipitation to analyze the hetero- dimerization of the AID C-terminal mutants with WT AID. The cotransfection and IP procedure were as described above (D).

various C-terminal mutants using a whole-cell extract. The results individual fractions of the glycerol gradient (Fig. 6). By applying showed that hnRNP K, hnRNP L, PABP, and hnRNP U were co- the reciprocal co-IP approach, we first determined the relative IPed with all of the C-terminal mutants as efficiently as with WT distributions of AID monomers and dimers in the gradient, fol- AID. HnRNP I and SERBP1 were also co-IPed with the C-ter- lowed by detection of hnRNP K and hnRNP L in the first 12 minal mutants but to a lesser degree. In particular, SERBP1 fractions, where they mostly migrated (Figs. 5B and 6A). Mono- showed much less association with the C-terminal mutant JP8B meric AID, found exclusively in fractions 3–4, was co-IPed with (Fig. S2C). These findings support our assumption that the broad hnRNP K whereas hnRNP K alone was quite heavily distributed mobility of AID was due to its complex formation with various through fractions 3–8. In contrast, hnRNP L began to be strongly RBPs. A portion of the AID dimer can be found in the HMW co-IPed with the AID from fraction 5 and tailed to fraction 12 in region probably because of their association with multiple RBPs. the LMW region. Thus, hnRNP K showed a preferential associ- We also examined whether AID and the above RBPs had the ation with the AID monomer whereas hnRNP L seemed to be same distribution profile in B cells. Intriguingly, endogenous or associated with the AID dimer (Fig. 6A). Because direct co-IP overexpressed AID in CH12F3-2A B cells appeared in the LMW analysis also confirmed that hnRNP K and hnRNP L interacted and HMW regions with a similar distribution as in HEK293T with the C-terminal AID mutants that existed only as monomers cells (Fig. S3). Moreover, the migration profiles of the RBPs also (Fig. S2C), the absence of hnRNP L at the monomeric AID matched well with those obtained using HEK293T cells (Fig. 5). fraction is likely due to the fact that the monomeric AID–hnRNP L complex contains additional proteins. Higher Order AID Complexes Contain Distinct RNPs. To examine We also examined whether the resolution of differential whether the IPed RBPs were associated with the monomeric complex formation of AID could be affected in a glycerol gra- or dimeric AID, we performed co-IP experiments with AID in dient run for a shorter duration (5 h). Interestingly, the shorter

E1548 | www.pnas.org/cgi/doi/10.1073/pnas.1601678113 Mondal et al. Downloaded by guest on September 23, 2021 PNAS PLUS

Fig. 3. The N terminus of AID is also required for AID dimerization. (A) Schematics of serially truncated N-terminal mutants of AID, in which the first 5, 10, and 26

amino acids were deleted. (B) Frequency histograms of the FACS analyses of HEK293T cells transfected with WT AID and N-terminally truncated mutants. BiFC INFLAMMATION IMMUNOLOGY AND constructs were cotransfected in pairwise combinations as indicated above each FACS profile. The Western blot analysis is shown for each AID expression profile, using tubulin as a loading control. (C) Percent MFI plot summarizing the results of the CB combination in the BiFC assay (mean ± SD; n = 3). The data from the other combinations are not shown because the CB combination produced the highest dimerization signal for WT AID. (D, Left) Representative FACS data showing the − − IgG1 switching efficiency of the N-terminally truncated AID mutants in AID / splenic B cells. Numbers indicate the percentage of IgG1 positive (+)cellsamong the GFP-gated population in each FACS profile. (Right) Relative frequencies of CSR and SHM of WT AID and its N-terminal mutants. (E, Left) BiFC analysis examining the heterodimeric interaction between WT AID and its N-terminal mutants. Frequency histograms of the FACS analysis of HEK293T cells transfected withthein- dicated pairwise combinations of BiFC constructs. (Right) Bar plots show the % MFI (mean ± SD; n = 3). (F and G) Immunoprecipitation analysis of the homo- and heterodimerization of N-terminal mutants by coexpressing CB combinations of BiFC constructs in HEK293T cells. AID was FLAG-tagged at the C terminus in the B construct. Cell lysates were IPed with anti-FLAG M2 agarose, and the coprecipitated proteins were analyzed by immunoblotting with an anti-AID antibody.

centrifugation revealed three BiFC peaks (I, II, and III) (Fig. dimer. We therefore generated a comprehensive overview of the 6C). Presumably, the latter two peaks merged into the HMW other newly identified AID-associated RBPs (hnRNP I, SERBP1, region in the longer run (Fig. 6B). PABP, hnRNP U, and hnRNP C) by running two types of glycerol The peak I at fractions 1–5 seems to contain the monomer only gradients (17 h and 5 h), followed by the co-IP analysis of each at fraction 1 and the monomer/dimer at fractions 2–5. The peak II fraction (Fig. 6 D and E). Because hnRNP Q and hnRNP E1 did at fractions 6–10 seems to contain mostly dimers complexed with not show any interaction with AID, they could not be detected in RBPs whereas peak III may consist of AID multimers/oligomers any of the fractions subjected to AID IP (Fig. S4 A and B). (Fig. 6 C and E). In general, RBP distributions after AID co-IP In parallel, we performed a control IP experiment using gra- were much narrower than those detected in the inputs, indicating dient fractions from HEK293T cells expressing no AID, which did that only small fractions of RBP form complexes with AID. In not show any specific IPed products (Fig. S2D). Taken together, particular, SERBP1 was found to be at fractions 1–10, indicating our findings indicate that the AID monomer and dimer associate that SERBP1 associates with the monomer as well as dimer of AID. with distinct sets of RBPs and RNAs, resulting in the broad hnRNP I, PABP, hnRNP U, and hnRNPC were largely found at distribution of AID in the LMW and HMW regions in glycerol peak II, indicating that these RBPs form complexes with the AID gradient sedimentation analysis.

Mondal et al. PNAS | Published online February 29, 2016 | E1549 Downloaded by guest on September 23, 2021 repair at the recombination phase of CSR. Given that DNA cleavage itself was not affected by the KD of hnRNP L, SERBP1, hnRNP U, or hnRNP I, these RNPs are likely to be involved at the postcleavage recombination phase of CSR. For example, hnRNP L was found to be involved in S–Ssynapsisregulation(18). Taken together, it can be envisaged that multiple RBP factors are required to execute different steps of DNA recombination. Most importantly, CSR-specific RBPs seem to associate with the AID dimer whereas hnRNP K associates with the AID monomer as well as C-terminal mutants that are proficient in DNA cleavage and SHM. These results further strengthen our view that the RNP complex formation specific to the higher order AID structures is distinctly involved in regulating DNA cleavage and recombination (Fig. 7E and Table S1). We also examined the gradient profiles, run under identical conditions, of the four APOBECs with their best interacting mKG combinations (Fig. S4). Unlike AID, all of the APOBEC family members showed single BiFC peaks that corresponded to their dimer positions. Although A3G showed a slightly broader distribution, the majority of the protein was located under the single BiFC peak (Fig. S4B). Strikingly, A2 produced a very sharp fluorescence peak that matched perfectly with its narrow protein distribution profile (Fig. S4A), which may indicate a higher self- interaction and a low association with other cellular proteins and RNAs in HEK293T cells. Discussion Based on the structural templates of A2 and A3G, several homology-modeling studies have postulated monomeric, dimeric, and tetrameric structures for AID (23, 25, 27, 43–46). However, the sequences of AID’s N and C termini do not match those of the APOBEC templates whose structures have been completely or partially resolved (27). Thus, the computational modeling studies are not informative for elucidating the molecular Fig. 4. Analysis of AID monomer and dimer by glycerol gradient sedimentation. basis of AID’s N- and C-terminal–specific functions in DNA (A) BiFC signal profile of the glycerol gradient fractions of the total cell extracts cleavage and recombination during SHM and CSR. Our BiFC of HEK293T cells transfected with the CB combination of BiFC constructs of WT or mutant AID, as indicated. A total of 23 fractions collected from the top to analysis of AID revealed that both of its termini are essential for bottom of the gradient were subjected to fluorescence intensity measurement. homodimer formation. Intriguingly, a head-to-tail oriented asso- Plot shows a representative dataset, and the positions of protein molecular ciation by BiFC showed the strongest fluorescence signal, which weight standards are indicated by open triangles. A mock fluorescence profile may give some insights into the AID dimeric structure, which has was obtained from HEK293T cells that were not transfected with any AID con- not yet been crystallized. Notably, the BiFC signals of AID and A1 struct. (B) Anti-AID immunoblots of the glycerol gradient fractions. (C) Anti-FLAG were much weaker than those of A2, A3G, and A3A, which and anti-HA IP of AID from the gradient fractions. Cell lysate was prepared from showed no construct (combination) dependency. It is thus possible HEK293T cells transfected with pairwise mKG-CB combinations of WT AID, that AID and A1 have a unique dimerization property. The where the C- and B-constructs harbored AID-Flag and AID-HA, respectively. Both weaker intrinsic dimerization property of AID may be particularly input and IPed fractions were analyzed by immunoblotting (IB) with an anti-AID beneficial for its ability to form multiple higher order structures antibody. Fractions enriched with monomers (mono) and dimers/multimers are through its interchangeable association with different functional indicated below the blots. (D) Anti-FLAG IP analysis of the gradient fractions cofactors. The BiFC technique itself may have stabilized the dimer derived from WT and mutant (P20 and JP8B) AID-expressing cells. HEK293T cells and helped us to detect the complexes with higher sensitivity. were transfected with the CB combination of BiFC constructs in which the Consistent with this possibility, the BiFC-positive AID dimer was B-construct harbored Flag-tagged AID. Both input and IPed fractions were found to be stable during glycerol gradient centrifugation and did analyzed by immunoblotting with an anti-AID antibody. not dissociate upon RNase or high-salt treatment. The AID IP products also contained a number of RBPs, in- Requirement of RNA-Binding Proteins for AID’s Functions. We pre- cluding hnRNPs that are known to form multiprotein complexes viously characterized hnRNP K and hnRNP L as AID cofactors with other proteins and RNAs. These binding partners partly ex- plain why AID forms higher order structures with broad mobilities for DNA cleavage and postcleavage recombination, respectively on the glycerol gradient sedimentation. Although we previously (18). We therefore examined whether CSR was affected by other identified hnRNP K and hnRNP L by the direct IP of AID, we newly identified AID-associated RBPs. Knockdown (KD) of could not reveal any relationship between AID’s structure and co- SERBP1 and hnRNP U in CH12F3-2A cells reduced the CSR in ’ A B factor association (18). The detection of AID s dimerization by accordance with their KD efficiencies (Fig. 7 and ). Less, but BiFC and the separation of monomers and dimers by glycerol significant, reduction of CSR was observed by KD of hnRNP I. gradient centrifugation enabled us to demonstrate functionally im- We then examined the effect of the KD of these RBPs on AID- portant RBP interactions with AID’s various structural states. We induced DNA cleavage. A ligation-mediated PCR (LM-PCR)- confirmed that AID’s association with RBPs was sensitive to RNase based double-stranded break (DSB) assay showed a significant and to high-salt treatment, suggesting involvement of RNA–protein decrease in the DNA-cleavage signal, especially in the case of interaction in complex formation. AID seems to form multimers SERBP1 and hnRNP U KD (Fig. 7 C and D). However, the that do not seem to interact with the functional RBPs identified. DNA-break signal became comparable with the control in the We showed that hnRNP K, previously identified as a putative presence of T4 polymerase, which blunts the DNA ends with high DNA-cleavage cofactor, preferentially associated with the mo- efficiency, suggesting that SERBP1 or hnRNP U deficiency causes nomeric AID. On the other hand, hnRNP L, which is involved in a defect in the DNA end processing that is required for DNA S–S synapse during CSR, associated with the AID dimer as well

E1550 | www.pnas.org/cgi/doi/10.1073/pnas.1601678113 Mondal et al. Downloaded by guest on September 23, 2021 as the monomer. The dimer fraction of AID was associated with PNAS PLUS several other critical RBP cofactors, which seem to be involved in CSR. Among those, hnRNP U and SERBP1, whose KD sig- nificantly affected CSR, were confirmed to be involved in the postbreak repair step required for recombination. Although hnRNP C and hnRNP M were detected in larger AID–RNP complexes, they did not seem to contribute to CSR function (18). Based on these findings, we favor the idea that the monomeric AID–hnRNP K complex is involved in DNA cleavage for SHM and CSR whereas the dimeric AID–RNP complex is involved in the CSR-associated recombination function, which absolutely requires the C terminus of AID. This scenario is consistent with the observation that C-terminal AID mutants are CSR-defective. Moreover, the requirement of both the N and C termini for stable dimer formation and the requirement of the N terminus for the associations with hnRNP K and hnRNP L indicate why both the N and C termini are critical for AID’s function. In fact, many AID mutants with N-terminal deletions in humans and mice are known to lose both CSR and SHM. In contrast, all of the C-terminal mutants that failed to form dimers retained their SHM activity, suggesting that monomeric AID (without the C terminus), which interacts with hnRNP K, is sufficient for the DNA-cleavage function. Furthermore, CSR seems to require the dimeric structure of AID, which forms larger complexes with hnRNP L, hnRNP U, hnRNP I, SERBP1, or other factors to fully execute recombination. Consistent with this view, the AID mutant G23S (47), which has compromised SHM but intact CSR activity, and the mutant S3A (48, 49), which has a higher CSR activity than WT, both had intact dimerization ability and also migrated to the HMW region upon glycerol gradient centrifugation (Fig. S5). Curiously, mutations at the putative RNA-binding motif (G133V and G133P) (50) resulted in loss of the BiFC signal and an altered association with the RBPs (Fig. S6) that are important for DNA cleavage and recombination for CSR. Despite their lesser expression level compared with WT AID, both G133V and G133P mutants showed a stronger associa- tion with hnRNP K and hnRNP L than with hnRNP U, hnRNP I, SERBP1, or PABP. In particular, these AID mutants showed a nearly complete loss of interaction with hnRNP U (Fig. S6C). These results suggested that these mutants are unable to form dimers or to interact with CSR-associated AID–RNP complexes. Because AID’s dimerization and RNP complex formation are critical for CSR, the CSR defect of these mutants can be explained by their inability to associate with the RBPs and RNA essential for CSR. It is critical to understand the functional properties of the AID INFLAMMATION

C terminus because its loss does not affect DNA cleavage but IMMUNOLOGY AND abolishes AID’s dimerization and CSR recombination function. The extreme C terminus of A1 is also important for dimerization; the A1 dimer associates with the cofactor A1CF and forms the functional APOB–mRNA editing complex (38, 39). A1 not only associates with A1CF or RBM47, but also interacts with other RBPsandhnRNPs(AB,Q,C,andK)(17,51–54). These cofactors regulate APOB mRNA editing, either positively or negatively, and IL8 mRNA stability (54). The functional relevance of all of the A1-associated RBPs is not yet fully understood, but they may participate in a variety of RNA-processing events by A1. In fact, large-scale sequence analyses by two independent groups recently revealed novel A1-specific C-to-U editing signatures in the UTR of a dozen mRNAs (55, 56). Therefore, the dimeric form of AID, like that of A1, may form an as-yet unidentified RNA- editing complex(es) in association with its CSR-specific RBP Fig. 5. RNA-dependent association of AID with RBPs. (A) Glycerol gradient (10–60% wt/vol) sedimentation analysis of the total cell extract of HEK293T cells transfected with the CB combination of BiFC constructs of WT AID. Cell extracts were treated with either 150 μg/mL RNase A or 500 mM NaCl and by immunoblotting (IB) with their corresponding antibodies. Indicated is the loaded onto the gradient. A total of 23 fractions were collected from the top presence (+) or absence (−) of AID and RNase A. (C) Co-IP analysis of the to the bottom of the gradient, followed by fluorescence intensity measure- association of RBPs with AID using the total cell extract of HEK293T cells ment of the fractions, which was plotted. The profiles of the mock and the WT transfected with the CB pair of WT AID BiFC constructs, in which the AID from Fig. 4A are included as reference. An anti-AID Western blot below B-construct harbored FLAG-tagged AID. Before the co-IP analysis, the cell the plot shows the distribution of AID in the fractions after RNase or salt lysates were either untreated or treated with RNase A or 500 mM NaCl. treatment. (B) Effect of AID expression and/or RNase A treatment on the FLAG-tagged AID was pulled down by anti-FLAG, and co-IPed proteins were glycerol gradient distribution of seven RNA-binding proteins (RBPs), analyzed analyzed by immunoblotting with RBP-specific antibodies, as indicated.

Mondal et al. PNAS | Published online February 29, 2016 | E1551 Downloaded by guest on September 23, 2021 Fig. 6. Monomeric and dimeric AID form RNP complexes. (A) Glycerol gradient (10–60% wt/vol) sedimentation analysis of the total cell extract of HEK293T cells transfected with the CB combination of BiFC constructs of WT AID, in which HA and FLAG were fused to the C and B construct, respectively. The gradient fractions (1–12) were subjected to IP by anti-FLAG or anti-HA, followed by immunoblot detection of AID, hnRNP K, and hnRNP L in the input and IPed fractions, as indicated Top and Right.(B and C) BiFC fluorescence intensity profiles of AID–AID interaction in the glycerol gradient fractions obtained from the 17-h and 5-h runs. (D and E) Analysis of the association of RBPs with AID in the fractions obtained after glycerol gradient (10–60% wt/vol) sedimentation of the total cell extract of HEK293T cells transfected with the CB BiFC constructs of WT AID, in which AID in the B-construct was fused to FLAG. Glycerol gradient sedimentation was performed for 17 h (D)and5h(E), and each fraction (1–23) was subjected to IP by anti-FLAG. Both the input and IPed fractions were subjected to immunoblotting with the indicated antibodies. Fractions containing different forms of AID are indicated by bars below the blots.

cofactors, such as hnRNP L, hnRNP U, SERBP1, and others. In- whether these RBPs are part of a single megacomplex or of isolated deed, we found that dimeric AID migrated to the HMW region complexes with AID because the loss of a potential factor by KD with distinct BiFC peaks and interacted with different RBPs. might affect the stability of the other components in an AID– Moreover, the identification of A1CF-like hnRNP cofactors for RNP complex. Nevertheless, we think that specific RBPs in dis- AID suggests that AID can form functional RNP complexes sim- tinct AID–RNP complexes assist AID in recombination-relevant ilar to A1. RNA editing, which may lead to generation of novel proteins Interestingly, A3A, which is known to mutate the genome involved in the S–S synapse and end-joining phases. Further heavily and is implicated in breast cancer development, was re- study will be required to fully explore the DNA-cleavage and cently found to edit specific RNAs in a cell type-specific manner recombination-specific AID–RNP complexes and their target (57, 58). Although the crystal structure study of A3A suggested a RNAs. It is also possible that AID possesses an as-yet unknown cooperative dimerization model for its action on DNA, the novel mode of translational regulation, by which a protein is up- structural basis of A3A’s RNA-editing and RNP complex- or down-modulated when its coding mRNA becomes bound to a forming abilities is still unknown (37, 59). specific AID–RNP complex. Because A1 and other APOBEC members have been sug- gested to exist also as monomers in cells, it is likely that AID Materials and Methods exists in an equilibrium between monomer and dimer. The Bimolecular Fluorescence Complementation Assay. The plasmids expressing low affinity of AID’s dimerization probably helps it transform the AID–mKG fusion constructs were transferred either individually or in dynamically from one state to another and thus to form distinct pairwise combinations (Fig. S1) into HEK293T cells in 12-well plates. FACS function-specific AID–RNP complexes. In the present study, we analysis was performed 48 h after transfection in a BD Biosciences FACSCa- libur cytometer. The mKG-BiFC–positive cells were detected in the FITC demonstrated that specific AID-associated RBPs have the po- ’ channel, and the data were analyzed in the live gate using CellQuest soft- tential to direct AID s function toward either DNA cleavage or ware (BD Biosciences). The mKG-BiFC assay was described previously (29, 33), recombination. Because CSR recombination can be regulated at and the stepwise details are also available in the manufacturer’s instruction – either the S S synapse or the DNA end-processing repair phase manual (Code No. AM-1100; MBL). (6, 11, 12), we speculate that at least two independent mRNAs are involved (7, 11, 60). Thus, it is reasonable to propose that distinct Glycerol Gradient Sedimentation Assay. HEK293T cells were plated on 10-cm RBPs are involved in AID–RNP complex formation and that their dishes and transfected with a pair of AID–mKG fusion constructs. In parallel, mock KD affects different stages of CSR. Currently, it is not clear transfection was performed to prepare the control cell extract. Forty-eight hours

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Fig. 7. CSR requires SERBP1 and hnRNP U at the DNA repair phase of CSR. (A) Representative FACS profiles of IgA switching in CH12F3-2A cells treated with the indicated siRNAs. Cont., control. Cells stimulated for IgA switching by CIT or left untreated are indicated by CIT(+) and CIT(−), respectively. The percentage of switched cells after 24 h of CIT stimulation is indicated in each FACS profile. (B) IgA switching and KD efficiency plots were generated from independent experiments. Immunoblots confirmed the specific KD of SERBP1and hnRNP U in CH12F3-2A cells. (C) AID-induced double-strand DNA break detection by the LM-PCR assay in CH12F3-2A cells treated with the indicated siRNAs. T4 polymerase-treated (T4+) and -untreated (T4−) DNA samples were subjected to Sμ-specific LM-PCR, followed by Southern blot hybridization with an Sμ-specific probe. Fourfold titrations of the input DNA were performed, and the

semiquantitative PCR of the GAPDH served as an internal control for the DNA samples used. (D) Quantitative representation of the LM-PCR signals INFLAMMATION generated, obtained by the densitometric analysis of all four lanes per sample. (E) A proposed model of AID’s structure–function relationship. AID monomers IMMUNOLOGY AND and dimers associate with a distinct set of RBPs and can form various higher order RNP structures containing yet unidentified RNAs. Such AID–RNP complexes are predicted to contribute uniquely in DNA cleavage and recombination.

after transfection, the cells were lysed in a lysis buffer [30 mM Tris·HCl (pH 7.4), fusion constructs. Cells were harvested 36 h after transfection and lysed 150 mM NaCl, 10% (vol/vol) glycerol, 1% Triton X-100, 0.05% Na-deoxy- as described above. Clarified cell lysates with an equal amount of total cholate, and 5 mM EDTA] supplemented with protease inhibitors protein were incubated with either anti-FLAG M2 agarose (Sigma) or (Roche). To analyze the effect of RNase A treatment, HEK293T cell ex- EZview Red anti-HA affinity gel (Sigma), using standard IP protocols. The IP tracts were preincubated at room temperature for 15 min with RNase A complex was washed three to four times in the same buffer containing 150 mM μ (130 g/mL). To analyze the effect of high-salt treatment, the cells were lysed NaCl. The bound proteins were eluted into SDS-sample buffer by boiling the with the lysis buffer containing 500 mM NaCl. Next, 1 mL of clarified cell beads. The eluted proteins were subjected to SDS/PAGE analysis and to im- extract was layered on top of an 11-mL 10–60% (wt/vol) glycerol gradient munoblotting with the specified antibodies. RNase- and high salt-treated prepared in the same lysis buffer using a Gradient Master. The gradient was cell extracts were prepared as described above, and the IP complex in centrifuged (40,000 rpm; 17 h or 5 h at 4 °C) in a P40ST rotor (Hitachi Ultra- each case was washed with buffer containing either RNase or 500 mM NaCl. centrifuge CP 70MX), and 0.5-mL fractions were collected in a BioComp gradient fractionator. The fluorescence intensity of each fraction was mea- Construction of mKG fused AID, CSR assay, gene knockdown in CH12F3-2A suredwithanEnVisionMultilabelPlateReader(Perkin-Elmer).Thefractions cells (62, 63), and DNA break analysis (64, 65) are described in SI Materials were also subjected to various analyses, such as immunodetection and co- and Methods. Antibodies used are listed in Table S2. IP. To analyze the endogenous AID and overexpressed AID in B cells, a CH12F3-2A clone was used that constitutively expresses an AID–GFP fusion ACKNOWLEDGMENTS. We thank Jin Highway and Keiko Yurimoto for protein. Cells were stimulated by CD40L, IL4, and TGFβ (CIT) for 24 h to induce excellent technical assistance in the mKG-BiFC work. We also thank Dr. Afzal endogenous AID expression (61). The cells were then lysed and subjected to Husain for support during the writing and for critical reading of the manuscript. This research was supported by Grant-in-aid for Specially Pro- gradient centrifugation and fraction collection, as described above. moted Research 17002015 (to T.H.) and Grant-in-Aid for Scientific Research 24590352 (to N.A.B.) from the Ministry of Education, Culture, Sports, Science, Immunoprecipitation (co-IP) from Total Cell Extract. HEK293T cells were and Technology of Japan. S.M. acknowledges support from the Human plated on six-well plates and transfected with the desired sets of AID–mKG Frontier Science Program (HFSP) for his postdoctoral fellowship.

Mondal et al. PNAS | Published online February 29, 2016 | E1553 Downloaded by guest on September 23, 2021 1. Muramatsu M, et al. (2000) Class switch recombination and hypermutation require 34. Huthoff H, Autore F, Gallois-Montbrun S, Fraternali F, Malim MH (2009) RNA- activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell dependent oligomerization of APOBEC3G is required for restriction of HIV-1. PLoS 102(5):553–563. Pathog 5(3):e1000330. 2. Honjo T, Nagaoka H, Shinkura R, Muramatsu M (2005) AID to overcome the limita- 35. Shlyakhtenko LS, et al. (2011) Atomic force microscopy studies provide direct evidence tions of genomic information. Nat Immunol 6(7):655–661. for dimerization of the HIV restriction factor APOBEC3G. JBiolChem286(5):3387–3395. 3. Faili A, et al. (2002) Induction of somatic hypermutation in immunoglobulin genes is 36. Shlyakhtenko LS, et al. (2013) Atomic force microscopy studies of APOBEC3G oligo- dependent on DNA polymerase iota. Nature 419(6910):944–947. merization and dynamics. J Struct Biol 184(2):217–225. 4. Petersen S, et al. (2001) AID is required to initiate Nbs1/gamma-H2AX focus formation 37. Bohn MF, et al. (2015) The ssDNA mutator APOBEC3A is regulated by cooperative and mutations at sites of class switching. Nature 414(6864):660–665. dimerization. Structure 23(5):903–911. 5. Wuerffel R, et al. (2007) S-S synapsis during class switch recombination is promoted by 38. Lau PP, Zhu HJ, Baldini A, Charnsangavej C, Chan L (1994) Dimeric structure of a distantly located transcriptional elements and activation-induced deaminase. Immunity human apolipoprotein B mRNA editing protein and cloning and chromosomal lo- – 27(5):711 722. calization of its gene. Proc Natl Acad Sci USA 91(18):8522–8526. 6. Boboila C, Alt FW, Schwer B (2012) Classical and alternative end-joining pathways for 39. Teng BB, et al. (1999) Mutational analysis of apolipoprotein B mRNA editing enzyme repair of lymphocyte-specific and general DNA double-strand breaks. Adv Immunol (APOBEC1): Structure-function relationships of RNA editing and dimerization. J Lipid – 116:1 49. Res 40(4):623–635. 7. Kato L, et al. (2012) An evolutionary view of the mechanism for immune and genome 40. Friew YN, Boyko V, Hu WS, Pathak VK (2009) Intracellular interactions between diversity. J Immunol 188(8):3559–3566. APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its 8. Ta VT, et al. (2003) AID mutant analyses indicate requirement for class-switch-specific association with RNA. Retrovirology 6:56. cofactors. Nat Immunol 4(9):843–848. 41. Ito S, et al. (2004) Activation-induced cytidine deaminase shuttles between nucleus 9. Barreto V, Reina-San-Martin B, Ramiro AR, McBride KM, Nussenzweig MC (2003) and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Proc Natl C-terminal deletion of AID uncouples class switch recombination from somatic hy- Acad Sci USA 101(7):1975–1980. permutation and gene conversion. Mol Cell 12(2):501–508. 42. Okazaki IM, et al. (2011) Histone chaperone Spt6 is required for class switch re- 10. Shinkura R, et al. (2004) Separate domains of AID are required for somatic hyper- combination but not somatic hypermutation. Proc Natl Acad Sci USA 108(19):7920–7925. and class-switch recombination. Nat Immunol 5(7):707–712. 43. Patenaude AM, et al. (2009) Active nuclear import and cytoplasmic retention of ac- 11. Sabouri S, et al. (2014) C-terminal region of activation-induced cytidine deaminase tivation-induced deaminase. Nat Struct Mol Biol 16(5):517–527. (AID) is required for efficient class switch recombination and gene conversion. Proc 44. Carpenter MA, Rajagurubandara E, Wijesinghe P, Bhagwat AS (2010) Determinants of Natl Acad Sci USA 111(6):2253–2258. – 12. Zahn A, et al. (2014) Activation induced deaminase C-terminal domain links DNA sequence-specificity within human AID and APOBEC3G. DNA Repair (Amst) 9(5):579 587. breaks to end protection and repair during class switch recombination. Proc Natl Acad 45. Mu Y, Prochnow C, Pham P, Chen XS, Goodman MF (2012) A structural basis for the Sci USA 111(11):E988–E997. biochemical behavior of activation-induced deaminase class-switch – 13. Jarmuz A, et al. (2002) An anthropoid-specific of orphan C to U RNA-editing recombination-defective hyper-IgM-2 mutants. J Biol Chem 287(33):28007 28016. enzymes on 22. Genomics 79(3):285–296. 46. Larijani M, Martin A (2012) The biochemistry of activation-induced deaminase and its – 14. Conticello SG, Langlois MA, Neuberger MS (2007) Insights into DNA deaminases. Nat physiological functions. Semin Immunol 24(4):255 263. Struct Mol Biol 14(1):7–9. 47. Wei M, et al. (2011) Mice carrying a knock-in mutation of Aicda resulting in a defect in 15. Mehta A, Kinter MT, Sherman NE, Driscoll DM (2000) Molecular cloning of -1 somatic hypermutation have impaired gut homeostasis and compromised mucosal complementation factor, a novel RNA-binding protein involved in the editing of defense. Nat Immunol 12(3):264–270. apolipoprotein B mRNA. Mol Cell Biol 20(5):1846–1854. 48. Gazumyan A, et al. (2011) Amino-terminal phosphorylation of activation-induced 16. Prohaska KM, Bennett RP, Salter JD, Smith HC (2014) The multifaceted roles of RNA cytidine deaminase suppresses c-myc/IgH translocation. Mol Cell Biol 31(3):442–449. binding in APOBEC cytidine deaminase functions. Wiley Interdiscip Rev RNA 5(4): 49. Honjo T, et al. (2012) The AID dilemma: Infection, or cancer? Adv Cancer Res 113:1–44. 493–508. 50. Zheng S, et al. (2015) Non-coding RNA generated following lariat debranching me- 17. Fossat N, et al. (2014) C to U RNA editing mediated by APOBEC1 requires RNA-binding diates targeting of AID to DNA. Cell 161(4):762–773. protein RBM47. EMBO Rep 15(8):903–910. 51. Lau PP, Zhu HJ, Nakamuta M, Chan L (1997) Cloning of an Apobec-1-binding protein 18. Hu W, Begum NA, Mondal S, Stanlie A, Honjo T (2015) Identification of DNA cleavage- that also interacts with apolipoprotein B mRNA and evidence for its involvement in and recombination-specific hnRNP cofactors for activation-induced cytidine de- RNA editing. J Biol Chem 272(3):1452–1455. aminase. Proc Natl Acad Sci USA 112(18):5791–5796. 52. Blanc V, et al. (2001) Identification of GRY-RBP as an apolipoprotein B RNA-binding 19. Conticello SG (2008) The AID/APOBEC family of nucleic acid mutators. Genome Biol protein that interacts with both apobec-1 and apobec-1 complementation factor to 9(6):229. modulate C to U editing. J Biol Chem 276(13):10272–10283. 20. Barreto VM, Magor BG (2011) Activation-induced cytidine deaminase structure and 53. Anant S, et al. (2001) Novel role for RNA-binding protein CUGBP2 in mammalian RNA functions: A species comparative view. Dev Comp Immunol 35(9):991–1007. editing: CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting 21. Navaratnam N, et al. (1998) cytidine deaminase provides a molecular with apobec-1 and ACF, the apobec-1 complementation factor. J Biol Chem 276(50): model for ApoB RNA editing and a mechanism for RNA substrate recognition. J Mol 47338–47351. – Biol 275(4):695 714. 54. Shimizu Y, et al. (2014) The RNA-editing enzyme APOBEC1 requires heterogeneous 22. Xie K, et al. (2004) The structure of a yeast RNA-editing deaminase provides insight nuclear ribonucleoprotein Q isoform 6 for efficient interaction with interleukin-8 into the fold and function of activation-induced deaminase and APOBEC-1. Proc Natl mRNA. J Biol Chem 289(38):26226–26238. – Acad Sci USA 101(21):8114 8119. 55. Rosenberg BR, Hamilton CE, Mwangi MM, Dewell S, Papavasiliou FN (2011) Tran- 23. Prochnow C, Bransteitter R, Klein MG, Goodman MF, Chen XS (2007) The APOBEC-2 scriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in crystal structure and functional implications for the deaminase AID. Nature 445(7126): transcript 3′ UTRs. Nat Struct Mol Biol 18(2):230–236. 447–451. 56. Blanc V, et al. (2014) Genome-wide identification and functional analysis of Apobec-1- 24. Krzysiak TC, Jung J, Thompson J, Baker D, Gronenborn AM (2012) APOBEC2 is a mediated C-to-U RNA editing in mouse small intestine and liver. Genome Biol 15(6):R79. monomer in solution: Implications for APOBEC3G models. Biochemistry 51(9):2008–2017. 57. Sharma S, et al. (2015) APOBEC3A cytidine deaminase induces RNA editing in 25. Brar SS, et al. (2008) Activation-induced deaminase, AID, is catalytically active as a monocytes and . Nat Commun 6:6881. monomer on single-stranded DNA. DNA Repair (Amst) 7(1):77–87. 58. Niavarani A, et al. (2015) APOBEC3A is implicated in a novel class of G-to-A mRNA 26. Bhagwat AS, Carpenter MA, Bujnicki JM (2008) Is AID a monomer in solution? DNA editing in WT1 transcripts. PLoS One 10(3):e0120089. Repair (Amst) 7(3):349–350, author reply 351–352. 59. Shlyakhtenko LS, Lushnikov AJ, Li M, Harris RS, Lyubchenko YL (2014) Interaction of 27. King JJ, et al. (2015) Catalytic pocket inaccessibility of activation-induced cytidine de- APOBEC3A with DNA assessed by atomic force microscopy. PLoS One 9(6):e99354. aminase is a safeguard against excessive mutagenic activity. Structure 23(4):615–627. 60. Doi T, et al. (2009) The C-terminal region of activation-induced cytidine deaminase is 28. Marianayagam NJ, Sunde M, Matthews JM (2004) The power of two: Protein di- responsible for a recombination function other than DNA cleavage in class switch merization in biology. Trends Biochem Sci 29(11):618–625. – 29. Kerppola TK (2006) Design and implementation of bimolecular fluorescence com- recombination. Proc Natl Acad Sci USA 106(8):2758 2763. + plementation (BiFC) assays for the visualization of protein interactions in living cells. 61. Nakamura M, et al. (1996) High frequency class switching of an IgM B lymphoma + – Nat Protoc 1(3):1278–1286. clone CH12F3 to IgA cells. Int Immunol 8(2):193 201. 30. Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a 62. Begum NA, Stanlie A, Nakata M, Akiyama H, Honjo T (2012) The histone chaperone probe of protein interactions in living cells. Annu Rev Biophys 37:465–487. Spt6 is required for activation-induced cytidine deaminase target determination – 31. Zhao W, et al. (2012) Nuclear to cytoplasmic translocation of heterogeneous nuclear through H3K4me3 regulation. J Biol Chem 287(39):32415 32429. ribonucleoprotein U enhances TLR-induced proinflammatory cytokine production by 63. Stanlie A, Aida M, Muramatsu M, Honjo T, Begum NA (2010) Histone3 lysine4 trime- stabilizing mRNAs in macrophages. J Immunol 188(7):3179–3187. thylation regulated by the facilitates chromatin transcription complex is critical for DNA 32. Chew TG, et al. (2013) A tudor domain protein SPINDLIN1 interacts with the mRNA- cleavage in class switch recombination. Proc Natl Acad Sci USA 107(51):22190–22195. binding protein SERBP1 and is involved in mouse oocyte meiotic resumption. PLoS 64. Schrader CE, Linehan EK, Mochegova SN, Woodland RT, Stavnezer J (2005) Inducible One 8(7):e69764. DNA breaks in Ig S regions are dependent on AID and UNG. J Exp Med 202(4):561–568. 33. Ueyama T, et al. (2008) Sequential binding of cytosolic Phox complex to phagosomes 65. Xu J, Husain A, Hu W, Honjo T, Kobayashi M (2014) APE1 is dispensable for S-region through regulated adaptor proteins: Evaluation using the novel monomeric Kusabira- cleavage but required for its repair in class switch recombination. Proc Natl Acad Green System and live imaging of phagocytosis. J Immunol 181(1):629–640. Sci USA 111(48):17242–17247.

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