RAG1相分离聚集对V(D)J基因重组的必要性

RAG介导的V(D)J基因重组是建立获得性免疫系统的基础。最新研究表明许多转录因子能够通过相分离(phase seperation)聚集。我们发现RAG1、RAG2以及NHEJ因子在细胞核内形成离散的聚合点；RAG1的K555A、E572A/D573A以及D584A/D585A突变体不能通过相分离而聚集；我们可以从细胞核中纯化出包含RAG1、RAG2以及NHEJ因子的巨大复合体(>20MDa)。我们假设RAG1和其他蛋白通过相分离聚集形成“V(D)J重组工厂”来完成重组反应。我们将(1)利用RAG1的K555A、E572A/D573A以及D584A/D585A突变基因敲入小鼠验证阻断RAG1相分离是否阻碍V(D)J重组；(2)研究NHEJ因子对RAG1相分离的影响；(3)阐明通过相分离和RAG1聚集的其他重要因子。这些结果将首次揭示RAG1相分离对细胞核内V(D)J基因重组的重要作用。

RAG mediated V(D)J recombination is the foundation for adaptive immune system. Recent studies proposed that many transcription factors organized into large complexes through phase separation. We found that RAG1, RAG2, and NHEJ factors are condensed at discrete nuclear foci；RAG1 K555A、E572A/D573A, and D584A/D585A mutants are unable to be incorporated into large complexes；we can purify maga complexes (>20 MDa) containing RAG1, RAG2, and NHEJ factors. We hypothesize that RAG1 and other proteins are assembled into V(D)J recombination machinery through phase separation mediated condensation to conduct recombination. We will (1) use RAG1 K555A, E572A/D573A, and D584A/D585A mutant gene knocked-in mice to determine if prevention of RAG1 phase separation affects V(D)J recombination; (2) determine if NHEJ factors affect RAG1 phase separation; (3) identify and characterize additional important factors that are associated with RAG1 during phase separation. These studies will elucidate first the important function of RAG1 phase separation in V(D)J recombination in the nuclei.