Interpretation | Xu Anlong (School of Life Sciences, Beijing University of Chinese Medicine)
The key mechanism that enables the human adaptive immune system to recognize diverse antigens and produce specific antibodies is the recombination activating genes RAG1 and RAG2 (collectively known as RAG), which encode the recombinase RAG that catalyzes V(D)J recombination to form diverse antibodies. In 1979, Japanese-American scientist Susumu Tonegawa (Nobel Laureate) and his team discovered this mechanism and proposed the transposon origin hypothesis of the rearrangement mechanism. Between 1988 and 1990, American scientist David Schatz (currently the chair of the Department of Immunobiology at Yale University) published several articles discovering the RAG (RAG1/RAG2) genes, and in 1998, he and Professor Martin Gellert from NIH simultaneously discovered that RAG has low-level transpositional activity in vitro, providing the first experimental evidence for the possibility of RAG-mediated transposition. However, the evolutionary context of the RAG molecule and the mechanisms of functional transformation have long remained unresolved.
In 2016, the journal Cell published a study led by my team on the evolution of the immune system, titled Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination, which provided direct evidence for the transposon origin hypothesis of RAG by discovering active ProtoRAG transposons in the genome of the invertebrate amphioxus, resolving nearly 40 years of debate on the origin of antibody rearrangement mechanisms and laying the foundation for further revealing how RAG evolved/tamed from transposase to recombinase through the relationship between primary structure and function.
Based on this, Dr. Zhang Yuhang, a postdoctoral researcher at Professor David Schatz’s lab at Yale University, studied the three-dimensional structure and function of the transposase ProtoRAG. In 2019, the journal Nature published Dr. Zhang’s research findings on ProtoRAG’s structure, which systematically elucidated the important molecular mechanisms by which RAG is tamed from transposase to recombinase through systematic studies of RAG and protoRAG and structural analysis of ProtoRAG, explaining how RAG forms significant functional differences by altering critical amino acids. As collaborators, our team was also deeply involved in this work, and I was one of the co-corresponding authors of this paper. In the same year, Dr. Liu Chang from Professor Schatz’s lab analyzed the structure of HzTransib, considered to be the ancestral gene of RAG1, further refining the evolutionary map of RAG molecules, and related research was also published in Nature. Our team’s research on the structure and function of the terminal repeat sequences of the protoRAG transposon was published in the journal National Science Review in 2020, providing new evidence for the evolution of RAG’s structure and function. To reveal how RAG function is negatively regulated, our team also identified a regulatory protein of the protoRAG transposon in amphioxus, and related research was published in Nature Communications in 2020.
Based on the above research, on October 21, 2021, Dr. Zhang Yuhang and Dr. Liu Chang published an article under the guidance of Professor David Schatz in Nature Reviews Immunology titled Structural insights into the evolution of the RAG recombinase, which systematically reviewed the functional evolution process of RAG from transposase to recombinase from a structural perspective, providing a systematic summary of the molecular evolutionary mechanisms of RAG. This review begins with the structural and functional differences of RAG and related RAG-like transposases, describing the structural changes and functional transformations that occurred during the evolution of RAG—how RAG was tamed from transposase to recombinase. It discusses various aspects, including changes in the DNA substrate-binding protease model, dynamics of the DNA-protease complex, mutations of single amino acid residues, and design of protease structural domains, illustrating how the RAG family has been tamed to remain and function in the eukaryotic genome.
The article proposes a model for RAG evolution starting from the Transib transposon, deeply reviewing the protein structures of HzTransib, ProtoRAG, and RAG, analyzing the precise cutting roles of the functional domains of RAG1 and RAG2 in cutting DNA substrates during the tetrameric structure, including coupled cutting and the 12/23 rule. Combining functional experiments, it proposes multiple mechanisms of transpositional function during the transition of RAG from transposase to recombinase, including single mutations (R848M, E649V) and the derivation of important functional domains, providing biochemical evidence, comparative biological evidence, and structural biological basis for the evolutionary model of RAG, offering structural biological insights and foundations for deeply understanding the origins and evolution of the rearrangement mechanisms that generate antibody/T cell receptor diversity.
This review systematically organizes important research on RAG evolutionary changes in recent years, suggesting that the RAG and transposase families can be tamed according to evolutionary needs. The emergence and evolution of RAG2 fully illustrate the role of evolutionary needs in protein derivation. In the process of RAG being tamed from transposon to recombinase, at least four mechanisms have preserved the endonuclease function while inhibiting the in vivo transpositional activity of RAG. However, many interesting questions remain unanswered, providing important references for follow-up research in this field.
Although the evolutionary mechanisms of RAG have been explained to some extent, there are still many interesting questions to be answered, and research continues… The Zhang Yuhang research group at Shanghai Jiao Tong University welcomes students interested in the following areas to join this research as postdoctoral fellows.
1. Research on the functional regulatory mechanisms in RAG molecular evolution;
2. Research on the molecular structure and catalytic mechanisms of RAG and related transposases.
https://www.nature.com/articles/s41577-021-00628-6
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