Activation-Induced Cytidine Deaminase Shuttles Between Nucleus and Cytoplasm Like Apolipoprotein B Mrna Editing Catalytic Polypeptide 1

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1

Satomi Ito, Hitoshi Nagaoka, Reiko Shinkura, Nasim Begum, Masamichi Muramatsu, Mikiyo Nakata, and Tasuku Honjo*

Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

Contributed by Tasuku Honjo, December 29, 2003 Activation-induced cytidine deaminase (AID) is a molecule central mRNA for a recombinase and mutator. However, the finding of to initiating class switch recombination, somatic hypermutation, DNA deaminase activity of AID led to the proposal that AID and gene conversion of Ig genes. However, its mechanism to deaminates cytosine (C) on a target DNA to generate a uracil initiate these genetic alterations is still unclear. AID can convert (U)–guanine(G) base pair that triggers base excision repair cytosine to uracil on either mRNA or DNA and is involved in DNA activities, thereby causing DNA cleavage and subsequent DNA cleavage. Although these events are expected to take place in the alterations (12–18). The additional supporting observation for nucleus, overexpressed AID was found predominantly in the cy- this hypothesis is that uracil DNA glycosylase deficiency affects toplasm. Here, we demonstrated that AID is a nucleocytoplasmic SHM base specificity and CSR efficiency (19, 20), which suggests shuttling protein with a bipartite nuclear localization signal and a involvement of U at some stage during the reaction. nuclear export signal in its N and C termini, respectively. In addition Sequestering a protein from its target in a different intracel- to previously identiﬁed genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic lular compartment is one of the general strategies to regulate polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the cellular activities that require interaction of many proteins. present ﬁnding provides another aspect to their resemblance, APOBEC1 was recently shown to shuttle between the nucleus and cytoplasm by association with importin at the N-terminal

suggesting that both may have homologous reaction mechanisms. IMMUNOLOGY nuclear localization signal (NLS) and the nuclear export ma- chinery at the C-terminal nuclear export signal (NES) of APO- he immune system has evolved specific mechanisms to BEC1 (21). APOBEC1 associates with and carries APOBEC1 Tdefend against numerous pathogens using a limited arsenal of Ig genes. After the formation of the primary Ig repertoire by complementation factor (ACF), which recognizes apoB100 V(D)J recombination of Ig genes during the developmental mRNA. ADAR2, the RNA editing adenosine deaminase that process, further diversification is achieved in antigen- acts on glutamate receptor pre-mRNA, accumulates in the experienced mature IgMϩ B cells by three types of genetic nucleolus but can shuttle to the nucleoplasm, where it edits the alterations, i.e., somatic hypermutation (SHM), gene conversion target pre-mRNA, suggesting that the ADAR2 activity can be (GC), and class switch recombination (CSR). SHM and GC modulated by functional sequestration from its substrate (22). introduce a large number of non-templated and templated point The intracellular localization of endogenous AID has not been mutations, respectively, in the Ig V region genes to raise extensively analyzed, because presently available reagents can- high-affinity antibodies after selection with a limited amount of not visualize the endogenous AID protein by immunostaining. antigen. CSR takes place between two S regions that locate 5Ј AID tagged by GFP revealed its predominant cytoplasmic adjacent to each Ig heavy chain constant (CH) region gene, distribution and raised the question of how cytoplasmic AID can resulting in replacement of the most upstream C␮ gene with reach its substrate in the nucleus, whether DNA or RNA (23). another downstream CH (C␥,C␧,orC␣) gene. B cells can thus Here we report that AID is a nucleocytoplasmic shuttling generate isotypes other than IgM, such as IgG, IgE, and IgA, protein with NLS and NES in its N and C termini, respectively. without changing antigen specificity (1). The finding adds another cellular biological similarity between Activation-induced cytidine deaminase (AID) is expressed AID and APOBEC1, suggesting their functional homology. almost exclusively in activated B cells (2). Disruption of the AID gene in mouse and human causes the hyper-IgM phenotype by Materials and Methods abolishing both SHM and CSR without any other signs of Retrovirus Constructs for GFP Fusion Proteins. The XhoI-NotI frag- lymphocyte dysfunction (3, 4). Furthermore, knockout of AID ment from pEGFP-N1 (Clontech) was cloned into the SalI-NotI in chicken B cell line DT40 also abolishes GC, which is spon- site of retrovirus expression vector pFB (Stratagene), designated taneously taking place in this cell line (5, 6). Inversely, ectopic pFB-GFP. Human AID (hAID) and its mutants were amplified expression of AID in non-B cells induces CSR and SHM (7–10). by PCR with use of Pyrobest, a high-fidelity DNA polymerase, These results indicate that AID is a molecule central to initiating ͞ CSR, SHM, and GC, the three types of Ig gene alterations that and cloned into EcoRI BamHI sites of pFB-GFP. Six amino acid occur in mature B lymphocytes. residues between the AID and GFP sequences, originated from Although AID is required for DNA cleavage (11), the detailed a residual multiple cloning sites of pEGFP-N1, are DPPVAT. mechanism by which AID induces these genetic events is still Deletion mutants were generated by PCR with internal primers under extensive debate. AID has the highest sequence homology that anneal where the truncations were introduced. with the apolipoprotein B (apoB) mRNA editing catalytic polypeptide 1 (APOBEC1), which edits a specific cytidine on Abbreviations: AID, activation-induced cytidine deaminase; wt, wild type; SHM, somatic mRNA of apoB100, a cholesterol carrier, converting it to mRNA hypermutation; CSR, class switch recombination; APOBEC1, apolipoprotein B mRNA edit- of apoB 48, a triglyceride carrier. AID, like APOBEC1, con- ing catalytic polypeptide 1; GC, gene conversion; NES, nuclear export signal; NLS, nuclear serves the catalytic motif of cytosine deaminase and has cytidine localization signal; ACF, APOBEC1 complementation factor; hAID, human AID; LMB, lep- deaminase activity on cytidine (2). These structural and func- tomycin B; PI, propidium iodide; tet, tetracycline. tional similarities between AID and APOBEC1 led to the *To whom correspondence should be addressed. E-mail: [email protected]. hypothesis that AID is an RNA editing enzyme that generates © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307335101 PNAS ͉ February 17, 2004 ͉ vol. 101 ͉ no. 7 ͉ 1975–1980 Downloaded by guest on September 29, 2021 Fig. 1. Subcellular localization of GFP-tagged AID and natural mutants. Expression vector for each GFP fusion of hAID wt (A), P20 (B), JP8B (C), JP41 (D), and GFP alone (E) was introduced by retrovirus system into NIH 3T3 cells. Confocal images of GFP signals with or without LMB treatment (10 ng͞ml for 150 min) are shown together with nuclear staining with PI of the same ﬁeld. The structure of each mutant is schematically illustrated (Left). Black boxes, NES; crosshatched boxes, NLS; hatched boxes, amino acid stretches caused by insertion or frame-shift. The 34- and 26-aa stretches inserted or replaced in P20 and JP8B are VTKPSTQFRRLSGPTDPQPRFEAIHSICFSLSLR and CMRLMTYETHFVLWDFDFDSNFQECHTR, respectively. (F) Amino acid sequences around NLS and NES regions of hAID, murine AID, and human APOBEC1 and NES consensus sequence are shown. Critical basic residues in NLS and conserved residues (L and F) in NES candidate are colored, and NES is underlined. Cytidine deaminase motif is indicated by a bar at the top of A. Numbers indicate residue positions.

Cell Culture and Virus Infection. NIH 3T3 cells were maintained in FACSCalibur with CELLQUEST software (Becton Dickinson). DMEM (Sigma) containing 10% FCS, 100 units͞ml penicillin, Dead cells were excluded from the analysis by forward scatter, and 100 ␮g͞ml streptomycin. Splenocytes from AIDϪ/Ϫ mice (3) side scatter, and PI gating. were cultured for 2 days in RPMI medium 1640 containing 10% FCS, 50 ␮M 2-mercaptoethanol, 2 mM L-glutamine, 100 Hypermutation Assay. As an artificial SHM target, a Dsred2 units͞ml penicillin, 100 ␮g͞ml streptomycin, 1 mM sodium (Clontech) expression cassette under the control of tetracycline pyruvate, 50 ␮g͞ml lipopolysaccharide (Sigma), and 15 ng͞ml (tet) inducible promoter was introduced into NIH 3T3 cells with mouse IL-4 (PeproTech, Boston). Splenocytes were preactivated a tet-off transactivator gene (8). Dsred2-expressing cells were for 2 days before infection. Cells were infected with pFB-hAID- enriched up to 95% by G418 selection and kept with tet GFP or mutant AID-GFP as described (24). thereafter. wt or mutant hAID-GFP was introduced by retrovirus vector to cells, and 4 days after infection tet was removed ϩ Immunofluorescence Microscopy. Cells were fixed in PLP solution from the culture medium. Thirteen days after tet removal, GFP cells were isolated by cell sorting with FACSVantage (Becton (2% paraformaldehyde͞10 mM NaIO ͞75 mM lysine͞37.5 mM ϩ 4 Dickinson) for DNA extraction. The purity of GFP cells after PBS) for 10 min. After permeabilization with 0.5% Triton X-100 the sorting was 88–95%. Dsred2 coding sequence was amplified in PBS for 5 min, cells were treated with 10 ␮g͞ml DNase-free by Pyrobest and cloned into pBSKS vector. Because of the ribonuclease (Nippon Gene, Toyama, Japan) for 120 min, and Ј ␮ ͞ GC-rich composition of the 5 region, only a part of the coding nuclei were stained with 1 g ml propidium iodide (PI; Sigma) sequence (base number 185–660 relative to the translation start for 10 min. In some experiments, cells were treated with 10 site) was determined and analyzed. ng͞ml leptomycin B (LMB), as described in the figure legends, before the fixation. LMB was provided from Dr. M. Yoshida Results (RIKEN Discovery Research Institute, Wako, Japan). Slides AID Is a Nucleocytoplasmic Shuttling Protein. To determine the were treated with the SlowFade Antifade kit (Molecular Probes) subcellular localization of AID in living cells, we have generated and were analyzed under an Axiophot 2 universal microscope an expression construct for hAID tagged with GFP at the C (Zeiss). The images were captured and processed by using an terminus (Fig. 1). The construct was introduced into NIH 3T3 MRC-1024 laser scanning confocal imaging system (Bio-Rad). cells by a retrovirus system because this cell line is competent for both CSR and SHM when AID and appropriate artificial Flow Cytometry. Cells were stained with biotinylated anti-mouse substrates were introduced together (7, 8). In agreement with the IgG1 (Pharmingen) and allophycocyanin–streptavidin (Vector). result in a human lymphoma cell line (23), hAID-GFP was Cells were analyzed for GFP and surface IgG1 expression by localized primarily in the cytoplasm (Fig. 1A Left).

1976 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307335101 Ito et al. Downloaded by guest on September 29, 2021 IMMUNOLOGY

Fig. 2. NES activity of the C-terminal 16 amino acid residues of hAID. Confocal images of GFP signals with or without LMB (10 ng͞ml for 150 min) are shown together with PI staining of the same ﬁeld: GFP fusion proteins of JP8Bdel (A), 196X (B), JP8B-C9 (C), JP8B-C16 (D), C9 (F), C16 (E), and L172A (G). C16 and C9 indicate C-terminal peptides of 16 and 9 amino acid residues, respectively. The structure of each mutant is shown (Left). Arrowhead, point mutation; horizontal arrow, replacement; X, termination; black box, NES; crosshatched box, NLS.

The cytoplasmic localization may represent either dynamic but taneous accumulation of JP8B in the nucleus is most likely due unbalanced shuttling from the nucleus or static inability in to the lack of the C-terminal 16 residues, and not to the addition entering the nucleus. To distinguish these two cases, we treated of missensed 26 amino acid residues by the frame-shift (Fig. 2A). the cells with LMB, which inhibits exportin1-dependent nuclear Another C-terminal deletion mutant, JP41 (190X)-GFP, also export (25). By this treatment, hAID-GFP changed its subcel- accumulated in the nucleus spontaneously (Fig. 1D). lular localization from the cytoplasm to the nucleus. After a Exportin1 recognizes a leucine-rich NES on a target protein 150-min incubation with LMB, the major GFP signal accumu- and exports it from the nucleus (27). The localization profiles of lated in the nucleus and only little remained in the cytoplasm hyper-IgM syndrome 2 AID mutants indicate the presence of a (Fig. 1A Right). The quick translocation of hAID-GFP strongly NES in the C-terminal region of AID. We checked the amino suggests that AID shuttles between the nucleus and cytoplasm acid composition of this region to see whether it matches the well and that its nuclear export pathway depends on exportin1. GFP documented NES motif. We found repeats of hydrophobic alone, however, distributed almost evenly in the nucleus and residues (leucine, valine, and phenylalanine) in every third to cytoplasm, and its profile was not sensitive to LMB (Fig. 1E). fourth interval with LXL (or LXF in mice) at the C-terminal end, which appeared to fit the NES motif (Fig. 1F) (28). To confirm AID Has NES in the C-Terminal Domain. We have shown that mutant the existence of NES, we constructed artificial mutants of the AIDs from hyper-IgM syndrome 2 patients are almost com- C-terminal domain. The deletion of C-terminal three and six pletely defective in CSR, but three mutants (P20, JP41, and residues (196X and 193X, respectively) resulted in the nuclear JP8B) that have replaced or truncated C-terminal 17 residues dominant pattern of GFP signal (Fig. 2B and data not shown), retain the SHM activity (26). To examine whether these muta- suggesting the three residues (LXL) at the C terminus of AID tions have any influence on the shuttling ability of AID, GFP are the essential part of the NES. We then examined whether the fusion proteins of these mutants were also introduced into NIH C-terminal region is sufficient for the nuclear export. Two 3T3 cells. The GFP fusion protein of P20, which has a 34-aa strategies were taken: addition of the C-terminal 16 residues insertion at residue 182 and is inactive for CSR but active for back to the JP8B or to the N terminus of GFP. The addition of SHM (26), showed LMB-dependent nuclear accumulation, es- the 16 residues restored cytoplasmic localization to both JP8B sentially the same as wt AID (Fig. 1B). Interestingly, JP8B (JP8B-C16) and GFP (C16-GFP), whereas the C-terminal nine carrying a frame-shift mutation at residue 183, which is almost residues did not (Fig. 2 C–F). The restored cytoplasmic proteins the same position as the P20 mutation, seemed to lose the responded to LMB, although the degree of nuclear accumulation shuttling ability and accumulated in the nucleus spontaneously was much higher for JP8B-C16 than for C16-GFP. (Fig. 1C). Because an artificial truncation at residue 183 (183X), There is another leucine-rich NES candidate at residues namely JP8Bdel, showed the same pattern as JP8B, the spon- 172–183 (Fig. 1F). Despite the marked similarity to the NES

Ito et al. PNAS ͉ February 17, 2004 ͉ vol. 101 ͉ no. 7 ͉ 1977 Downloaded by guest on September 29, 2021 Fig. 3. N-terminal NLS activity of hAID. Subcellular localizations of N-terminal truncation mutants ⌬N26hAID (A), ⌬N20hAID (B), ⌬N10JP8Bdel (C), and ⌬N5JP8Bdel (D) and point mutated natural mutants P7 (E) and P13 (F) as well as GFP protein fused with the N-terminal peptides (G) are shown with PI staining. The structure of each mutant is shown (Left). Arrowhead, point mutation; X, termination; black box, NES; crosshatched box, NLS. LMB treatments were done at 10 ng͞ml for 150 min.

consensus, point mutations of these leucines did not give any Both P7 and P13 are functionally null mutants for both CSR and indications of the NES activity (Fig. 2G and data not shown). SHM (26). However, P7 has a significant deaminase activity in Taking all of these observations together, we conclude that AID Escherichia coli, whereas P13 has no such activity, although its has a functional NES within the C-terminal 16 residues 183–198. deaminase motif is intact (data not shown), suggesting that protein conformational change is associated with the P13 mu- AID Has a Bipartite NLS in the N-Terminal Domain. AID was shown tation. To examine whether the bipartite NLS of AID is active to shuttle between the cytoplasm and nucleus, but how does it for an unrelated protein, we added the N-terminal 29 residues of enter the nucleus? As described above, JP8B-C16 responded to AID to the N terminus of GFP. The nuclear GFP level was LMB much more quickly and completely than did C16-GFP. increased by the addition of NLS (Fig. 3G vs. Fig. 1E). These Because GFP itself has no specific localization signal, it is more results indicate that AID has a functional bipartite NLS in the likely that JP8B has a specific signal for active import to the N terminus, which works efficiently in a correctly folded AID. nucleus. From an alignment with other APOBEC1-related sequences (21, 29), it appeared that AID has a putative bipartite Nucleocytoplasmic Shuttling of AID in B Lymphocytes. To examine NLS, two clusters of basic residues, at residues 8–25 (Fig. 1F). whether the subcellular localization of AID is regulated in the To analyze the putative NLS activity, we constructed N-terminal same manner in B lymphocytes as in fibroblasts, we infected region mutants of AID tagged with GFP. A mutant with deletion AIDϪ/Ϫ spleen B lymphocytes with wt and mutant hAID-GFP- of residues 2–26, ⌬N26hAID-GFP lost the capacity to enter the expressing retroviruses. wt hAID-GFP was localized in the nucleus, as shown by blocking nuclear export with the LMB cytoplasm and translocated into the nucleus upon the LMB treatment (Fig. 3A). The result is consistent with the notion that treatment, whereas ⌬N26hAID-GFP and P13-GFP were local- the putative NLS region is indeed functional. ized in the cytoplasm regardless of the LMB treatment (Fig. 4 NLS activity is not restored by adding back the N-terminal 6 A–C). Mutants lacking the C-terminal NES (JP8Bdel, 196X, and or 16 residues, as shown with ⌬N20hAID-GFP and JP8B) accumulated in the nucleus without LMB (Fig. 4 D–F). ⌬N10JP8Bdel-GFP (Fig. 3 B and C). On the other hand, a Therefore, we conclude that the localization of AID is regulated mutant with deletion of residues 2–5, ⌬N5-JP8Bdel-GFP, is similarly in B lymphocytes and in fibroblasts. transported into the nucleus (Fig. 3D). Consistently, a point mutation at residue arginine 24, namely P7, showed impaired Relevance of the Localization Signals to SHM and CSR. Analyses of nuclear transport (Fig. 1E). P13 with one point mutation at P20, JP41, and JP8B mutants have shown that the C-terminal 17 residue methionine 139 also appeared to be LMB-insensitive. amino acid residues of AID are critical for CSR but not for SHM

1978 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307335101 Ito et al. Downloaded by guest on September 29, 2021 suggests that the efficient export from the nucleus is not critical for induction of SHM. To determine whether the most C-terminal three residues (which are essential residues as NES but are dispensable for SHM) are relevant to CSR activity, we introduced 196X-GFP into lipopolysaccharide- and IL-4 stimulated spleen B cells of AIDϪ/Ϫ mice (3). On day 3, CSR efficiency in 196X-GFP- infected cells was slightly higher than background (0.35% IgGϩ) but very low (3–6% IgGϩ) compared with wtAID-GFP (38– 47% IgGϩ). We conclude that the three residues at the C terminus are critical for effective induction of CSR. Because it is likely that AID edits its target (RNA or DNA) in the nucleus (29), the defect of NLS may result in the total loss of the AID activity. In fact, a point mutation at residue arginine 24, namely P7, causes a complete loss of nuclear localization and function (26). Similarly, the NLS deletion mutants (⌬2–10, ⌬11–20, and ⌬2–20) of mouse AID completely lost their CSR and SHM activities together with nuclear localization activity (unpublished data). We conclude that the NLS region is critical for the AID function for CSR and SHM. Discussion AID is thought to deaminate C on either RNA or DNA to initiate the Ig gene alteration reactions (1, 18). Because not only DNA editing but also RNA editing are thought to take place in the nucleus (29), AID is likely to function in the nucleus. In

support of this view, AID fused with the hormone binding IMMUNOLOGY domain of the human estrogen receptor (AID-ER) quickly migrates to the nuclear fraction in concomitance with CSR induction after addition of the estrogen analogue that activates AID-ER by dissociating it from hsp90 (24). However, the AID-GFP fusion protein was previously demonstrated to local- ize in the cytoplasm (23). Here we have shown that AID has NLS and NES and can thereby dynamically shuttle between the nucleus and cytoplasm. The predominant cytoplasmic localization pattern of AID-GFP represents the biased balance to the Fig. 4. B lymphocytes show intracellular AID-GFP patterns similar to those of export from the nucleus, because the blockade of a nuclear ﬁbroblasts. Selected wt (A) and mutant AID-GFP representative for normal, export pathway evokes its rapid and robust accumulation in the disturbed import (B and C) or export (D–F) were introduced by retrovirus nucleus. The predominance of the nuclear export does not seem vector to preactivated AIDϪ/Ϫ B lymphocytes. Photos were taken 3 days after to be specific to the cell type, because the fibroblasts, B infection. LMB treatments were done at 10 ng͞ml for 150 min. lymphocytes, and human embryonic kidney cell line showed essentially the same pattern (Figs. 1A and 4A and data not shown). The nuclear localization of AID appears to be essential and GC (26, 30). Because this C-terminal region well overlaps to its function, because all of the NLS mutations tested so far lost with the NES, we examined SHM activity of GFP fusion of NES their activities for CSR and SHM in mammalian cells. deleted mutants, for which we used NIH 3T3 cells carrying a The role of NES signal may be 2-fold. First, the amount of AID Dsred2 reporter gene driven by the tet inducible promoter. in the nucleus at the steady state must be tightly controlled to When AID is expressed in NIH 3T3, SHM accumulates on the maintain a minimal level, because excessive amounts of nuclear actively transcribed reporter gene (8). As shown in the Table 1, AID may induce unregulated SHM not only in Ig genes (Table all GFP fusion proteins with NES deletion exhibited SHM 1) but also in non-Ig genes (31). Second, according to the RNA activity with higher frequencies than wt AID. In addition, it is editing hypothesis, edited mRNA in the AID–cofactor complex indicated that the N-terminal five residues are dispensable for should be transported to the cytoplasm for translation. In case SHM because ⌬N5-JP8Bdel-GFP is active in SHM. The result of APOBEC1, the majority of edited ApoB100 mRNA are

Table 1. Mutation frequency induced by mutant AID-GFP fusion protein P values versus* Clone Mutation Total Frequency Sequence mutated͞total (del. or ins.) bases per 104 None hAID

196X 7͞10 16 (1) 4,760 33.6 Ͻ0.001 Ͻ0.001 193X 5͞10 12 (0) 4,760 25.2 Ͻ0.001 0.006 JP8Bdel 8͞10 20 (3) 4,730 42.2 Ͻ0.001 Ͻ0.001 ⌬N5JP8Bdel 7͞10 20 (1) 4,714 42.4 Ͻ0.001 Ͻ0.001 hAID 7͞33 12 (2) 15,679 7.7 0.005 None 1͞27 1 (0) 12,851 0.8

*Fisher’s exact test for mutation͞bases. del., deletion; ins., insertion.

Ito et al. PNAS ͉ February 17, 2004 ͉ vol. 101 ͉ no. 7 ͉ 1979 Downloaded by guest on September 29, 2021 exported to the cytoplasm as a complex associated with APO- complex formation of APOBEC1 and ACF is essential not only BEC1 and ACF to avoid non-sense mediated decay (21). for target recognition but also for modulation of the intracellular Several possibilities can be raised to explain why NES mutants localization. Moreover, glycerol gradient sedimentation analysis showed higher mutator activities on the artificial substrate. One revealed that the active nuclear editing complex of APOBEC1 may speculate that AID requires specific cofactors not only for and ACF differs from that of the inactive cytoplasmic com- CSR but also for SHM. Cofactors for CSR and SHM may plex (34). compete for association with the AID molecule. Because asso- AID and APOBEC1 are known to have genetic and structural ciation of the CSR specific cofactor requires the C-terminal 17 homology that indicates their evolutionary relationship, namely residues of AID (26, 30), which overlaps with NES, the C- chromosomal locus proximity and conservation of essential terminal NES mutants can associate only with the SHM cofactor, motifs (2, 29, 35). Both AID and APOBEC1 are known to giving rise to enhanced SHM activities. Because P20 lost CSR function as a complex containing the homodimer and another activity specifically despite normal nuclear export, the presence protein for recognition of target (26, 34, 36, 37). apoB mRNA of NES is not sufficient for CSR activity, indicating that inter- edited by APOBEC1 has to be translated to manifest its phys- action of AID with the CSR-specific cofactor may require a iological function. Likewise, DNA cleavage activity of AID larger region than NES. Another possibility, which does not depends on de novo protein synthesis (ref. 24 and unpublished exclude the possiblity mentioned above, is that longer retention data). The present cellular biological findings that both APO- of AID in the nucleus may increase the chance to meet its BEC1 and AID are nuclear-cytoplasmic shuttling proteins con- substrate, either pre-mRNA or DNA. taining NLS and NES at their N- and C-terminal regions, Like AID, APOBEC1 shuttles between the nucleus and respectively, strengthens their similarity. Such extensive similar- cytoplasm and has both bipartite NLS and NES (21). Overex- ities between AID and APOBEC1 favor the hypothesis that they pressed APOBEC1 localizes in the nucleus and cytoplasm, and share functional homology, i.e., RNA editing. the preference is variable among cell types (21, 32, 33). ACF We thank Dr. M. Yoshida for providing LMB, Ms. Y. Sasaki for excellent expressed alone localizes almost exclusively in the cytoplasm technical assistance, Drs. S. Fagarasan and K. Kinoshita for critical (21), but ACF is imported into the nucleus when coexpressed reading of the manuscript, and Ms. K. Saito for preparation of the with APOBEC1 in a single cell. The NES activity of APOBEC1 manuscript. This work was supported by a Center of Excellence Grant is also sensitive to LMB. These observations suggest that a from the Ministry of Education, Science, Sports, and Culture of Japan.

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