产生抗体重排机制的原始RAG转座子的功能与演化研究

Origin of V(D)J recombination is an important question in immunology and evolutionary biology. In 2016, the applicant published a paper in Cell as the co-first author to report the discovery of ProtoRAG transposon in lancelet, which confirms V(D)J recombination was descended from RAG transposon. However, questions that what roles does the ProtoRAG play in amphioxus, and how does the ProtoRAG transposase evolve into the RAG recombinase exclusively for V(D)J recombination, become desiderated to be answered. Study on the transposition activity has revealed that insertions of ProtoRAG in lancelet genome were polymorphic among individuals, and efficiency of TIR substrates cleavage and recombination were highly dependent on TIR sequence. So, it was hypothesized that besides for the transposition activity of ProtoRAG in amphioxus, they might induce genome recombination by cleaving the TIR-like sequences in lancelet genome. Based on the newly discovered ProtoRAG, this project would study its transposition mechanism, recognition rules of TIR sequence, mechanism of transcription regulation, the possible roles in generation of lancelet genome diversity, and further reveal the evolution discipline of V(D)J recombination. Based on the transposition mechanism of ProtoRAG, this project would further improve the activity of ProtoRAG transposase and optimize the transposition system, then it would provide a new strategy for developing new genetic tools.

V(D)J抗体重排机制的起源是免疫学和进化生物学的重要科学问题。申请人于2016年以共同第一作者在Cell上发表论文报道了文昌鱼ProtoRAG转座子的发现，证实V(D)J重排起源于RAG转座子。但关于RAG转座子在文昌鱼中发挥怎样的功能，以及在进化过程中如何演化为负责抗体重排的RAG重组酶是有待回答的重要科学问题。转座活性研究发现RAG转座子的基因组位置具有个体多样性；其切割底物和介导重组的效率受TIR的显著影响，推测其可能切割基因组中TIR类似序列从而增加基因组多样性。本申请拟在发现ProtoRAG的基础上，进一步对其转座活性，转座机制，识别TIR序列的规则，转录调控机制，是否参与基因组多样性产生等问题开展研究，以期进一步揭示V(D)J抗体重排机制的演化规律。本研究还将在ProtoRAG转座机制的基础上，进一步优化RAG转座酶的活性和转座体系，将为开发新的基因遗传操作工具提供新的技术。

RAG重组酶在V(D)J抗体重排中发挥关键核心作用，它的出现被认为是脊椎动物适应性免疫起源的重要里程碑。前期研究中，我们在无脊椎动物文昌鱼中发现的ProtoRAG转座子为RAG重组酶起源于转座子的假说提供了最直接证据。为进一步揭示原始RAG转座子的功能与演化，我们对ProtoRAG发挥功能依赖的TIR序列规则，宿主调控ProtoRAG表达和功能的机制以及RAG转座子的起源等重要问题开展深入研究并取得系列成果。首先，我们系统地阐明了ProtoRAG转座子发挥活性所依赖的TIR序列组成规则，它们与脊椎动物RAG重组酶依赖的12/23RSS具有相似的结构组成特征，该发现佐证了RSS序列与RAG转座子TIR序列有共同起源。其次，我们揭示了BbYY1通过多种方式调控ProtoRAG表达和功能的机制，包括：BbYY1抑制ProtoRAG编码的BbRAG1L/2L基因转录，BbYY1与TIR中TR5基序结合帮助BbRAG1L/2L更准确定位TIR和精确切割DNA以及BbYY1抑制ProtoRAG转座。这些发现增加了我们对RAG转座子如何在宿主中长期存活和被驯化成为重组酶的理解。最后，我们首次在真核生物的多个低等进化分支中发现RAG同源基因片段，并从单细胞真核藻类——Aureococcus anophagefferens中克隆得到目前最古老的RAG转座子，表明早在10多亿年以前，RAG转座子就已经出现。同时，我们也提出真核生物RAG与Transib转座子共同起源于细菌Transib转座子并在真核生物时期发生分化的新观点。因此，本项目研究成果显著的推进了对原始RAG转座子功能与演化的认知，也为后续开发并应用RAG介导基因重组的技术提供了技术基础。