VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity

Abstract

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineagespecific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J H rearrangement, whereas V H -DJ H and V j -J j rearrangements are severely impaired. D-J H coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination. The EMBO Journal (2012) 31, 945-958.

VprBP Is Required for Efficient Editing and Selection of Igκ+ B Cells, but Is Dispensable for Igλ+ and Marginal Zone B Cell Maturation and Selection

“…1A), and showed that B cell development was arrested at the pro B-to-pre B cell transition in these animals (3). Concomitant expression of functionally rearranged anti-HEL Ig heavy and light chain transgenes could partially bypass the developmental block, arguing that VprBP plays a role in V(D)J recombination, but evidence for defects in cell cycle progression and increased apoptosis in pre-B cells from these animals pointed to potential additional roles for VprBP in cell proliferation and survival (3). In several other genetic models of B cell developmental arrest, enforced Bcl2 expression has been shown to enable some maturing B cells to bypass the developmental block (4–7).…”

“…How the RAG1 amino-terminus promotes this outcome remains unclear, but evidence from our laboratory and others suggest it functions at least in part as a protein interaction domain to recruit factors to facilitate chromosomal V(D)J recombination. We recently identified that Vpr binding protein (VprBP; DCAF1), a substrate adaptor molecule for the Cul4-Roc1-DDB1 (CRL4) and EDD/UBR5 E3 ubiquitin ligase complexes (2), associates with the amino-terminal region of RAG1, and that VprBP is required for B cell development and plays a role in V(D)J recombination (3). Specifically, we found that conditional disruption of Vprbp early in B cell development arrests B cell maturation at the pro B-to-pre-B cell transition, but this developmental block is partially rescued by expressing functionally rearranged Ig transgenes.…”

“…Specifically, we found that conditional disruption of Vprbp early in B cell development arrests B cell maturation at the pro B-to-pre-B cell transition, but this developmental block is partially rescued by expressing functionally rearranged Ig transgenes. Loss of VprBP expression in B cells is associated with impaired V H -DJ H gene rearrangement, reduced fidelity of V H -DJ H joining, defects in cell cycle progression, and increased apoptosis (3). …”

Histone methylation and V(D)J recombination

“…Importantly, mutations in the non-core regions cause SCID, but their functions are not well understood [24]. The RAG1 N terminus in the non-core region contains a RING finger domain which has ubiquitin ligase (E3) activity itself or when in complex with VprBP/DDB1/Cul4A/Roc1 proteins [25][26][27]. It was reported that this region binds and ubiquitinates histone H3, possibly linking the RAG complex to the DSB repair process.…”

A First-in-Class NAE Inhibitor, MLN4924, Blocks Lentiviral Infection in Myeloid Cells by Disrupting Neddylation-Dependent Vpx-Mediated SAMHD1 Degradation

“…The CRL4(DCAF1) E3 ligase has been shown to be essential for DNA replication, cell cycle regulation, and embryonic development (10)(11)(12). Its ubiquitination capacity was found to be usurped by different proteins of diverse viruses (13,14).…”