The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes. Mus musculus

Activation-induced cytidine deaminase (AID) mediates class switching by binding to a small fraction of single-stranded DNA (ssDNA) to diversify the antibody repertoire. The precise mechanism for highly selective AID targeting in the genome has remained elusive. Here, we report an RNA-binding protein, ROD1 (also known as PTBP3), that is both required and sufficient to define AID-binding sites genome-wide in activated B cells. ROD1 interacts with AID via an ultraconserved loop, which proves to be critical for the recruitment of AID to ssDNA using bi-directionally transcribed nascent RNAs as stepping stones. Strikingly, AID-specific mutations identified in human patients with hyper-IgM syndrome type 2 (HIGM2) completely disrupt the AID interacting surface with ROD1, thereby abolishing the recruitment of AID to immunoglobulin (Ig) loci. Together, our results suggest that bi-directionally transcribed RNA traps the RNA binding protein ROD1, which serves as a guiding system for AID to load onto specific genomic loci to induce DNA rearrangement during immune responses. Overall design: ROD1/PTBP3 CLIP-seq was performed with naïve splenic B cells and LPS-activated B cells, and two biological replicates were labeled as Rep1 and Rep2. Two biological repeats of AID CLIP-seq in LPS-activated B cells were labeled as Rep1 and Rep2; in addition, two AID CLIP-seq libraries were also generated upon the depletion of ROD1/PTBP3. For comparison, IgG antibodies were applied to generate two replicates of CLIP-seq libraries for negative control purposes. To examine whether ROD1/PTBP3 ablation alter the global nascent RNA levels, two biological repeats of GRO-seq were performed on LPS-activated B cells.