Abstract
The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.
Highlights
During their differentiation from hematopoietic stem cells, B and T lymphocytes assemble antigen receptor genes from arrays of Variable, Diverse, and Joining segments through a process termed V(D)J recombination
We show here that ectopically-expressed murine RAG1 is turned over in a human, non-lymphoid cell line in a manner sensitive to either CRL or proteasome inhibition or loss of Viral protein r Binding Protein (VprBP), suggesting widespread expression and evolutionary conservation of factors involved in RAG1 degradation
In previous work [30], we demonstrated that VprBP (DCAF1) controls RAG1 turnover in murine B cells
Summary
During their differentiation from hematopoietic stem cells, B and T lymphocytes assemble antigen receptor genes from arrays of Variable, Diverse, and Joining segments through a process termed V(D)J recombination. This process requires the recombination activating gene proteins (RAG1 and RAG2), which together initiate V(D)J recombination by catalyzing the formation of DNA double-strand breaks (DBSs) at sites immediately adjacent to antigen receptor gene segments. The DNA ends are processed and re-ligated by the non-homologous end-joining pathway to form functional immunoglobulin (Ig) or T cell receptor variable genes in B and T lymphocytes, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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