Vertebrate immune systems can respond to many infections and insults. This ability relies on a diverse binding repertoire of antigen receptors. Antigen receptor diversity is created through a process called V(D)J recombination in which arrayed gene segments are shuffled to form functional receptors. This process introduces breaks in chromosomal DNA catalyzed by the RAG1-RAG2 protein complex and requires strict regulation to guard genome integrity. Here we demonstrate a mode of RAG1 regulation by nucleolar sequestration. RAG1’s nucleolar localization is dynamically regulated and is disrupted during a B cell’s transition to a prorecombination state, leading to increased recombination.
V(D)J recombination assembles and diversifies Ig and T cell receptor genes in developing B and T lymphocytes. The reaction is initiated by the RAG1-RAG2 protein complex which binds and cleaves at discrete gene segments in the antigen receptor loci. To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full-length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar proteins in a manner dependent on amino acids 216 to 383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increased V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.