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

Juan Chen,Meizhu Bai,Jinsong Li,Xiaohua Yu,Yingpeng Yao,Yang Yu,Ruibao Su,Yuanchao Xue,Baidong Hou,Chao Zhang,Di Wang,Xihao Hu,Rong Ye,Zhaokui Cai,Shuyang Yu,Changchang Cao,Lei Wang

doi:10.1038/s41422-018-0076-9

Abstract

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.

Highlights

This RNA-guided system prompted us to consider a possibility that a similar strategy might be naturally employed in activated B cells to impart Activation-induced cytidine deaminase (AID) specificity via newly transcribed RNAs, which would be in line with the observation that the glutathione S-transferase (GST)-AID fusion protein is more efficiently cross-linked by UV to RNA than DNA.[8]
We performed a λN/BoxB tethering assay,[37] in which multiple BoxB elements were inserted into RNA generated from a reporter and AID was fused to λN to recognize those BoxB elements, thereby forcing AID to newly synthesized RNA in HEK293 cells
ROD1 directs AID targeting genome wide Since both ROD1 and AID are required for class switch recombination (CSR) and they form a complex in vivo, we examined whether ROD1 regulates the genome-wide targeting of AID

Summary

Introduction

Once activated in germinal centers, B cells express AID to initiate somatic hypermutation (SHM) in exons in variable regions, promoting affinity maturation.[2,3] In the meantime, these cells evoke class switch recombination (CSR) between the Cμ constant exon and one of the downstream exons, such as Cγ, Cα, or Cε, to produce high-affinity IgG, IgA, or IgE antibodies.[2,4,5] AID, an enzyme that deaminates cytidines in single-stranded DNA (ssDNA) hotspots,[6,7,8,9] is primarily loaded onto immunoglobulin (Ig) loci to induce double-strand breaks or mutations.[10,11] Motifs enriched in AID hotspots are RGYW/WRCY (where R = purine, Y = pyrimidine, and W = A or T),[9] which in theory appear once every 36 bp in the human genome. The presence of such motifs in the genome is not sufficient for AID binding both in vitro and in vivo.[12,13]

Methods

Results

Conclusion