Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase

Abstract

Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to function as an RNAediting enzyme, based on its strong homology with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1), the enzyme that edits apolipoprotein B-100 mRNA in the presence of the APOBEC cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Because A1CF is structurally similar to heterogeneous nuclear ribonucleoproteins (hnRNPs), we investigated the involvement of several well-known hnRNPs in AID function by using siRNA knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated disruption. We found that hnRNP K deficiency inhibited DNA cleavage and thereby induced both CSR and SHM, whereas hnRNP L deficiency inhibited only CSR and somewhat enhanced SHM. Interestingly, both hnRNPs exhibited RNA-dependent interactions with AID, and mutant forms of these proteins containing deletions in the RNA-recognition motif failed to rescue CSR. Thus, our study suggests that hnRNP K and hnRNP L may serve as A1CF-like cofactors in AID-mediated CSR and SHM.class switch recombination | somatic hypermutation | activation-induced cytidine deaminase | B cell | IgH A ntigen-stimulated mature B cells express activation-induced cytidine deaminase (AID), an essential enzyme for somatic hypermutation (SHM) and class switch recombination (CSR) at the Ig locus (1, 2). AID induces DNA breaks at the variable (V) and switch (S) regions during SHM and CSR, respectively. Most of the mutations produced during SHM are introduced by errorprone DNA synthesis during single-strand break (SSB) repair (3, 4). In contrast, CSR requires the conversion of SSBs to doublestrand breaks (DSBs), followed by recombination between two DSB ends located in the donor and acceptor S regions (5, 6). The entire process of CSR is accomplished through elaborate DNArepair processes involving S-S synapse formation and end-joining.The mechanism by which AID functions differently in DNA cleavage and recombination at different loci remains unclear. Functional studies of a large number of AID mutants revealed that the N-terminal AID mutations impair SHM and CSR, indicating that the AID N terminus, which also possesses a bipartite nuclear-localization signal, is required for DNA cleavage in both SHM and CSR (7-9). On the other hand, C-terminal AID mutations suppressed the recombination activity of CSR but had no effect on SHM, indicating that the C terminus of AID, which contains a nuclear-export signal, is required for the recombination activity associated specifically with CSR (7, 9, 10). Indeed, recent studies showed that defects in the AID C terminus compromise DNA end-joining and S-S synapse formation without perturbing DNA breakage at either the V or S region (11, 12), also suggesting a specific role for the AID C terminus in the recombination step of CSR. Given AID's small size (198 resi...

Enhanced Content of IgG in Burkitt’s Lymphoma Cells after Treatment with the Topoisomerase II Inhibitor, Lucanthone

“…Recent studies have implicated hn RNP's as cofactors for AID [18]. Their role, if any, in lucanthone's effects need further consideration.…”

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

“…We next examined the distribution profiles of AID-interacting RBPs (hnRNP I/PTBP1, SERBP1, PABP, hnRNP U, and hnRNP C) 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.…”

“…Requirement of RNA-Binding Proteins for AID's Functions. We previously characterized hnRNP K and hnRNP L as AID cofactors for DNA cleavage and postcleavage recombination, respectively (18). We therefore examined whether CSR was affected by other newly identified AID-associated RBPs.…”

“…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-S synapsis regulation (18). Taken together, it can be envisaged that multiple RBP factors are required to execute different steps of DNA recombination.…”

“…The APOB mRNA editing ability of A1 is highly dependent on its cofactors, A1CF/ACF (15,16) and RBM47 (17), both of which belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Recently, two A1CF-like hnRNPs, hnRNP K and hnRNP L, were identified as the cofactors of AID and found to be involved in the cleavage and recombination of DNA, respectively (18). Because the N and C termini of AID differentially regulate two functions of AID-cleavage and recombination, respectivelywe speculated that the AID termini would be critical for functioncoupled cofactor association.…”