Abstract
The assembly of immunoglobulin genes in developing B lymphocytes by V(D)J recombination is initiated by the RAG1–RAG2 endonuclease complex. We previously identified an interaction between RAG1 and viral protein R binding protein (VprBP) (also known as DNA damage binding protein 1 cullin 4–associated factor 1 [DCAF1]), a substrate receptor for the cullin 4–really interesting new gene (RING) E3 ubiquitin ligase (CRL4). In this dissertation, we report that B cell–intrinsic loss of VprBP increases RAG1 protein levels and disrupts expression of the endoribonuclease Dicer, which is essential for microRNA maturation. Rag1/2 transcription is known to be derepressed by loss of microRNA-mediated suppression of phosphatase and tensin homolog, raising the possibility that the elevated level of RAG1 observed in VprBP-deficient B cells is caused indirectly by the loss of Dicer. However, we show that VprBP restrains RAG1 expression posttranscriptionally and independently of Dicer. Interestingly, in addition to its role in negative regulation of RAG1 expression, we identify a secondary role for VprBP in transcriptional activation of Rag expression.| Loss of VprBP stabilizes RAG1 protein, which we show is normally degraded via a mechanism requiring both 20S proteasome and cullin–RING E3 ubiquitin ligase activity. Furthermore, we show that RAG1 stabilization through small molecule inhibition of cullin–RING E3 ubiquitin ligase activation promotes V(D)J recombination in a murine pre–B cell line. Interestingly, despite the requirement for CRL4 E3 ligase activity in RAG1 turnover, RAG1 degradation does not depend on ubiquitylation of RAG1. Thus, in addition to identifying a role for VprBP in maintaining Dicer levels in B cells, our findings reveal the basis for RAG1 turnover and provide new insight into how developing B cells prevent excessive RAG activity.