Proximity-based proteomic profiling uncovers distinct interactome of human RAG1 and RAG2

Abstract

The recombination-activating gene (RAG) complex initiates adaptive immunity by catalyzing V(D)J recombination to generate diverse antigen receptors. While the catalytic function of the RAG core is well defined, its regulatory interactions and physiological roles remain poorly understood due to limited knowledge of RAG-associated proteins. The RAG complex forms a heterotetramer of two RAG1 and RAG2 subunits, yet the individual contributions of each subunit remain unclear. Here, we use TurboID-mediated proximity labelling to map the human RAG interactome. By fusing TurboID to RAG1 or RAG2, we identify 88 RAG1- and 146 RAG2-associated proteins, with only 23 shared proteins, indicating distinct sets of proximal proteins. Although RAG1 and RAG2 are thought to exert their physiological functions by forming a complex, they display distinct potential interaction networks, suggesting subunit-specific functions and revealing their spatial proximity to each subunit. These findings uncover distinct RAG1 and RAG2 interaction landscapes and establish a framework for exploring broader RAG functions in immunity.

1. Introduction

The recombination-activating gene (RAG) complex is a core component of the adaptive immune system, playing a pivotal role in generating antigen receptor diversity [1–3]. RAG specifically recognizes recombination signal sequences (RSS) that flank variable (V), diversity (D), and joining (J) gene segments in immunoglobulin (Ig) and T-cell receptor (TCR) loci [2–4]. It catalyzes site-specific DNA cleavage at RSS-coding sequence junctions, producing double-strand breaks (DSBs) that initiate V(D)J recombination and enable the assembly of a diverse antigen receptor repertoire [3,5] (Supplementary Figure S1). Evolutionary and biochemical analyses suggest that RAG originates from a transposon-encoded transposase capable of cut-and-paste transposition of mobile genetic elements [6–12]. In jawed vertebrates, this function became increasingly specialized: while RAG lost its transposase activity, it retained DNA endonuclease activity—a shift likely reflecting co-evolution with the host genome and the emergence of a more tightly-regulated immune repertoire [11,13,14]. Despite decades of investigation into the enzymatic and structural properties of RAG, its regulation within the complex cellular environment remains poorly understood [15,16]. This gap limits our understanding of RAG’s broader biological roles, especially in humans, including how its activity is modulated in different contexts and whether it may participate in functions beyond V(D)J recombination.

Beyond its catalytic activity, RAG interacts with various host factors to coordinate its function with subsequent DNA repair. For instance, RAG binds to modified histones, influencing chromatin accessibility during V(D)J recombination [17–20]. The RING domain of RAG1 also functions as an E3 ubiquitin ligase, thereby mediating histone ubiquitination and modulating chromatin dynamics [21–24]. Additionally, RAG1 interacts with Vpr-binding protein (VprBP; also known as DCAF1, DDB1- and CUL4-associated factor 1), which targets RAG1 for ubiquitin-mediated degradation, thereby controlling the levels of V(D)J recombination activity under physiological conditions [25,26]. RAG has also been reported to associate with Ku70, a key factor in the non-homologous end joining (NHEJ) DNA repair pathway, to promote efficient DSB repair during V(D)J recombination [27]. While these studies have identified individual RAG-associated factors, the full extent of the RAG interaction network remains elusive. This is likely due to the transient and dynamic nature of many RAG interactions that are hard to capture with conventional biochemical approaches. As a result, comprehensive mapping of the RAG interactome is still lacking.