The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Abstract

The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.activation-induced deaminase ͉ antibody diversification ͉ immunoglobulin class switching ͉ exportin ͉ nuclear transport A ctivation-induced deaminase (AID) plays a central role in antibody gene-diversification, triggering both somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating deoxycytidine residues within the Ig locus to yield deoxyuridine (1, 2).AID is largely found in the cytoplasm of activated B cells but shuttles between cytoplasm and nucleus (3-6). The C-terminal amino acids of AID comprise a nuclear-export sequence (NES): removal of this NES causes AID to be restricted to the nucleus, whereas its addition to a heterologous nuclear protein drives export of the fusion protein into the cytoplasm (3-5). The sequence of the AID C-terminus conforms well to the consensus established for substrates of the Crm1-dependent export machinery: in keeping with this, export mediated by the AID NES is sensitive to leptomycin B.Analysis of both clinical and contrived mutations in AID have revealed that the AID C-terminal region is also essential for its function in CSR, although not in SHM (7-9). The C-terminal mutations that interfere with AID's ability to potentiate CSR do not compromise its ability to deaminate deoxycytidine in biochemical or bacterial genetic assays. Thus, as previously suggested by others, either the nuclear/cytoplasmic shuttling of AID must be specifically required for CSR or, alternatively, factors specific for CSR must interact with the C-terminal region of AID in an area overlapping the NES (4, 5, 7-9).It was with a view to discriminating these possibilities that we embarked on a refined mutagenic dissection of the AID NES and investigated the ability of heterologous NESs to substitute for the AID NES. Our results suggest that although nuclear export is likely necessary for CSR, this is not the sole function of the AID C-terminal region, with the data raising the possibility that successful CSR depends on the precise quality of AID binding to exportin or a related protein. Results NES Function and CSR Correlate in Alanine-Scanning Mutagenesis.To ascertain whether there was a correlation between export activity and CSR, we performed an alanine-scanning mut...

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

“…Microhomology-mediated end-joining also could be the result of impaired recruitment of cofactor(s) involved in the processing/recombination of AID-induced lesions. Several studies have strongly suggested that the C-terminal part of AID could recruit a repair cofactor or target AID to specific areas, such as nuclear pores, thereby providing a special DNA repair/recombination environment (32,33,39). The detection of DSBs in the Sμ region from AID +/C-termΔ B cells supports the impaired recruitment hypothesis for the AID +/C-termΔ group.…”

“…This discrepancy could be related to the sequencing of different regions in Sμ, or to the fact that the murine study analyzed mutations in germline (i.e., unrearranged) Sμ regions from B cells undergoing in vitro CSR, whereas we analyzed rearranged Sμ regions from AID +/C-termΔ patients that were successfully switched in vivo. A recent study suggested that AID participates via its C-terminal part in a multimolecular complex (supposedly at nuclear pores) necessary for its stability and CSR (33). Thus, the lower frequency in the Sμ core region from Sμ-Sα junctions derived from UNG2 −/− and AID +/C-termΔ B cells could be explained by differences in the DNA repair/recombination environment.…”

Cytoplasmic activation-induced cytidine deaminase (AID) exists in stoichiometric complex with translation elongation factor 1α (eEF1A)

“…To investigate the possible involvement of AID's specific NES in the eEF1A interaction, we asked whether the interaction was retained with a multiply mutated NES mutant (M8) that nevertheless confers nuclear export function and protein stabilization (15). The results reveal that AID's NES can be substantially altered without seriously jeopardizing the eEF1A interaction (Fig.…”

“…The N-terminal portion of AID therefore appears necessary (either indirectly or directly) for the eEF1A interaction. The interpretation of the result with chimera E is slightly less straightforward because the removal of the C-terminal region of AID [which also contains its nuclear export sequence (NES) as well as residues implicated in AID's cytosolic retention] results in AID destabilization (9,12,15).…”

AID-induced T-lymphoma or B-leukemia/lymphoma in a mouse BMT model

“…It must be noted that expression levels of mouse mutant D189-198 as well as human mutants JP8B and P20 in GFP-sorted BM cells were lower than those of WTs, possibly due to protein instability of these C-terminal mutants. 30 Therefore, we cannot answer the question whether CSR activity is indispensable for oncogenicity of AID, but we assume that the maximum ability of AID to cause lymphoma requires an intact form of AID with SHM activity.…”