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

Abstract

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structurespecific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions., which is expressed in antigen-stimulated mature B cells, is essential for Ig somatic hypermutation (SHM) and class-switch recombination (CSR) (1, 2). AID induces DNA breaks at the variable (V) and switch (S) regions during SHM and CSR, respectively (3, 4). Although both processes are initiated by AID-induced DNA cleavage, point mutations at the V region are executed mostly by error-prone DNA repair whereas CSR is accomplished by recombination of cleaved ends at donor and acceptor S regions (5, 6). However, the detailed mechanisms by which AID carries out the two mechanistically distinct functions for SHM and CSR have yet to be uncovered (7). Studies on AID mutants revealed that AID's N-and C-terminal domains are distinctly required for its DNA-cleavage and recombination functions, respectively (8-10). Mutations at the N terminus of AID impair SHM as well as CSR whereas those at the C terminus abrogate CSR only and show increased SHM activity. Recent studies demonstrated that the CSR process after DNA cleavage, including the synapsis formation between cleaved ends, is impaired with the C-terminally defective AID, indicating that AID's C terminus confers a CSR-specific recombination function, independent of AID's DNA cleavage function, by an unknown mechanism (11,12).AID belongs to the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide) family of cytidine deaminases (CDDs) and shows high sequence homology with APOBEC1 (A1) (1,13,14), which edits apolipoprotein B (APOB) mRNA. 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...

Antibody repertoire and gene expression dynamics of diverse human B cell states during affinity maturation

“…However, we detected little enrichment of our class switch recombination signature in non-cycling DZ GC B cells (Figure 3L), and although there was an enrichment of class switch recombination genes in cycling B cell populations, this likely reflects their involvement in cell cycle-linked DNA recombination and repair. Our analysis also highlights many other genes previously linked with class switch recombination (Figure 3M), including those capable of binding to switch region sequences within the IgH locus ( NME2, NCL, DDX21 ), interacting with the class switch recombination machinery ( NPM1, SERBP1 ) or regulating AICDA /AICDA transcript/protein stability ( mir155HG, HSP90AB1 ) (Borggrefe et al, 1998, Hanakahi et al, 1997, Mondal et al, 2016, Orthwein et al, 2010, Shinozaki et al, 2006, McRae et al, 2017, Zheng et al, 2019). Notably, the microRNA gene miR155HG , formerly called BIC (B-cell Integration Cluster), was up-regulated in preGC B cells and has been shown to be essential for B cells to form GCs and undergo class switch recombination in mice (Thai et al, 2007, Vigorito et al, 2007).…”

AID Recognizes Structured DNA for Class Switch Recombination

“…It should be noted that AID.mono only cooperatively oligomerizes on G4, but not on a simpler structured substrate, such as branched DNA, that may be formed by local secondary structures (Figure 4B). The AID oligomerization may be further facilitated by the CTT (Mondal et al, 2016) and result in previously observed processivity (Pham et al, 2003). …”

Dimerisation of APOBEC1 is dispensable for its RNA/DNA editing activity and modulates its availability

“…Based on the increased levels of A1-GFP either after treatment with the MG-132 proteasomal inhibitor or through coexpression of APOBEC1 ( Figure 4B and C), the more straightforward interpretation for such opposite effect is the existence of a correlation between dimerisation of APOBEC1 and its degradation. Similarly to another family member, AID, which is predominantly degraded in the nucleus through the proteasome (75), and whose monomeric and dimeric forms interact with different molecules (76), it could be possible that APOBEC1 dimers are preferentially degraded through their interaction with a factor, be it nucleic acids or proteins, that triggers their degradation. Alternatively the dimer could induce changes in its interactors: e.g.…”