The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line

• Published in: PloS one Vol. 16; no. 10; p. e0258683
• Main Authors: Schabla, N. Max, Swanson, Patrick C.
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 14.10.2021; Public Library of Science (PLoS)
• Subjects: Adaptor proteins; Antigens; Biology and Life Sciences; Cell cycle; Degradation; Endonuclease; Enzymes; Genes; Immunology; Localization; Lymphocytes; Lymphocytes B; Lymphocytes T; Medicine and Health Sciences; Plasmids; Proteasomes; Proteins; RAG1 protein; RAG2 protein; Research and analysis methods; Substrates; T cell receptors; Ubiquitin; Ubiquitin-protein ligase; V(D)J recombination; United States > US; Nebraska
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0258683

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 CRL4 VPRBP/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.