Recombination-activating gene

The recombination-activating genes (RAGs) encode parts of a protein complex that plays important roles in the rearrangement and recombination of the genes encoding immunoglobulin and T cell receptor molecules. There are two recombination-activating genes RAG1 and RAG2, whose cellular expression is restricted to lymphocytes during their developmental stages. The enzymes encoded by these genes, RAG-1 and RAG-2, are essential to the generation of mature B cells and T cells, two types of lymphocyte that are crucial components of the adaptive immune system.^[1]

recombination-activating gene 1
Identifiers
Symbol	RAG1
NCBI gene	5896
HGNC	9831
OMIM	179615
RefSeq	NM_000448
UniProt	P15918
Other data
Locus	Chr. 11 p13
Search for Structures Swiss-model Domains InterPro

recombination-activating gene 2
Identifiers
Symbol	RAG2
NCBI gene	5897
HGNC	9832
OMIM	179616
RefSeq	NM_000536
UniProt	P55895
Other data
Locus	Chr. 11 p13
Search for Structures Swiss-model Domains InterPro

Recombination-activating protein 2
Identifiers
Symbol	RAG2
Pfam	PF03089
InterPro	IPR004321
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Recombination-activating protein 1
Identifiers
Symbol	RAG1
Pfam	PF12940
InterPro	IPR004321
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Function

In the vertebrate immune system, each antibody is customized to attack one particular antigen (foreign proteins and carbohydrates) without attacking the body itself. The human genome has at most 30,000 genes, and yet it generates millions of different antibodies, which allows it to be able to respond to invasion from millions of different antigens. The immune system generates this diversity of antibodies by shuffling, cutting and recombining a few hundred genes (the VDJ genes) to create millions of permutations, in a process called V(D)J recombination.^[1] RAG-1 and RAG-2 are proteins at the ends of VDJ genes that separate, shuffle, and rejoin the VDJ genes. This shuffling takes place inside B cells and T cells during their maturation.

RAG enzymes work as a multi-subunit complex to induce cleavage of a single double stranded DNA (dsDNA) molecule between the antigen receptor coding segment and a flanking recombination signal sequence (RSS). They do this in two steps. They initially introduce a ‘nick’ in the 5' (upstream) end of the RSS heptamer (a conserved region of 7 nucleotides) that is adjacent to the coding sequence, leaving behind a specific biochemical structure on this region of DNA: a 3'-hydroxyl (OH) group at the coding end and a 5'-phosphate (PO₄) group at the RSS end. The next step couples these chemical groups, binding the OH-group (on the coding end) to the PO₄-group (that is sitting between the RSS and the gene segment on the opposite strand). This produces a 5'-phosphorylated double-stranded break at the RSS and a covalently closed hairpin at the coding end. The RAG proteins remain at these junctions until other enzymes (notably, TDT) repair the DNA breaks.

The RAG proteins initiate V(D)J recombination, which is essential for the maturation of pre-B and pre-T cells. Activated mature B cells also possess two other remarkable, RAG-independent phenomena of manipulating their own DNA: so-called class-switch recombination (AKA isotype switching) and somatic hypermutation (AKA affinity maturation).^[2] Current studies have indicated that RAG-1 and RAG-2 must work in a synergistic manner to activate VDJ recombination. RAG-1 was shown to inefficiently induce recombination activity of the VDJ genes when isolated and transfected into fibroblast samples. When RAG-1 was cotransfected with RAG-2, recombination frequency increased by a 1000-fold.^[3] This finding has fostered the newly revised theory that RAG genes may not only assist in VDJ recombination, but rather, directly induce the recombinations of the VDJ genes.

Structure

As with many enzymes, RAG proteins are fairly large. For example, mouse RAG-1 contains 1040 amino acids and mouse RAG-2 contains 527 amino acids. The enzymatic activity of the RAG proteins is concentrated largely in a core region; Residues 384–1008 of RAG-1 and residues 1–387 of RAG-2 retain most of the DNA cleavage activity. The RAG-1 core contains three acidic residues (D₆₀₀, D₇₀₈, and E₉₆₂) in what is called the DDE motif, the major active site for DNA cleavage. These residues are critical for nicking the DNA strand and for forming the DNA hairpin. Residues 384–454 of RAG-1 comprise a nonamer-binding region (NBR) that specifically binds the conserved monomer (9 nucleotides) of the RSS and the central domain (amino acids 528–760) of RAG-1 binds specifically to the RSS heptamer. The core region of RAG-2 is predicted to form a six-bladed beta-propeller structure that appears less specific than RAG-1 for its target.

Cryo-electron microscopy structures of the synaptic RAG complexes reveal a closed dimer conformation with generation of new intermolecular interactions between two RAG1-RAG2 monomers upon DNA binding, compared to the Apo-RAG complex which constitutes as an open conformation.^[4] Both RAG1 molecules in the closed dimer are involved in the cooperative binding of the 12-RSS and 23-RSS intermediates with base specific interactions in the heptamer of the signal end. The first base of the heptamer in the signal end is flipped out to avoid the clash in the active center. Each coding end of the nicked-RSS intermediate is stabilized exclusively by one RAG1-RAG2 monomer with non-specific protein-DNA interactions. The coding end is highly distorted with one base flipped out from the DNA duplex in the active center, which facilitates the hairpin formation by a potential two-metal ion catalytic mechanism. The 12-RSS and 23-RSS intermediates are highly bent and asymmetrically bound to the synaptic RAG complex with the nonamer binding domain dimer tilts towards the nonamer of the 12-RSS but away from the nonamer of the 23-RSS, which emphasizes the 12/23 rule. Two HMGB1 molecules bind at each side of 12-RSS and 23-RSS to stabilize the highly bent RSSs. These structures elaborate the molecular mechanisms for DNA recognition, catalysis and the unique synapsis underlying the 12/23 rule, provide new insights into the RAG-associated human diseases, and represent a most complete set of complexes in the catalytic pathways of any DDE family recombinases, transposases or integrases.

Evolution

Based on core sequence homology, it is believed that RAG1 evolved from a transposase from the Transib superfamily.^[5] No Transib family members include an N-terminal sequence found in RAG1 suggesting the N-terminal of RAG1 came from a separate element. The N-terminal region of RAG1 has been found in the transposable element N-RAG-TP in the sea slug, Aplysia californica, which contains the entire RAG1 N-terminal.^[6] It is likely that the full RAG1 structure was derived from the recombination between a Transib and the N-RAG-TP transposon.^[7]

A transposon with RAG2 arranged next to RAG1 has been identified in the purple sea urchin.^[8] Active Transib transposons with both RAG1 and RAG2 ("ProtoRAG") has been discovered in B. belcheri (Chinese lancelet) and Psectrotarsia flava (a moth).^[9][10] The terminal inverted repeats (TIR) in lancet ProtoRAG have a heptamer-spacer-nonamer structure similar to that of RSS, but the moth ProtoRAG lacks a nonamer. The nonamer-binding regions and the nonamer sequences of lancet ProtoRAG and animal RAG are different enough to not recognize each other.^[9] The structure of the lancet protoRAG has been solved (PDB: 6b40​), providing some understanding on what changes lead to the domestication of RAG genes.^[11]

Although the transposon origins of these genes are well-established, there is still no consensus on when the ancestral RAG1/2 locus became present in the vertebrate genome. Because agnathans (a class of jawless fish) lack a core RAG1 element, it was traditionally assumed that RAG1 invaded after the agnathan/gnathostome split 1001 to 590 million years ago (MYA).^[12] However, the core sequence of RAG1 has been identified in the echinoderm Strongylocentrotus purpuratus (purple sea urchin),^[13] the amphioxi Branchiostoma floridae (Florida lancelet).^[14] Sequences with homology to RAG1 have also been identified in Lytechinus veriegatus (green sea urchin), Patiria minata (sea star),^[8] the mollusk Aplysia californica,^[15] and protostomes including oysters, mussels, ribbon worms, and the non-bilaterian cnidarians.^[16] These findings indicate that the Transib family transposon invaded multiple times in non-vertebrate species, and invaded the ancestral jawed vertebrate genome about 500 MYA.^[8] It is hypothesized that the absence of RAG-like genes in jawed vertebrates and urochordates^[16] is due to horizontal gene transfer or gene loss in certain phylogenetic groups due to conventional vertical transmission.^[13] Recent analysis has shown the RAG phylogeny to be gradual and directional, suggesting an evolutionary path that relies on vertical transmission.^[16] This hypothesis suggests that the RAG1/2-like pair may have been present in its current form in most metazoan lineages and was lost in the jawed vertebrate and urochordate lineages.^[7] There is no evidence that the V(D)J recombination system arose earlier than the vertebrate lineage.^[7] It is currently hypothesized that the invasion of RAG1/2 is the most important evolutionary event in terms of shaping the gnathostome adaptive immune system vs. the agnathan variable lymphocyte receptor system.

Selective pressure

It is still unclear what forces led to the development of a RAG1/2-mediated immune system exclusively in jawed vertebrates and not in any invertebrate species that also acquired the RAG1/2-containing transposon. Current hypotheses include two whole-genome duplication events in vertebrates,^[17] which would provide the genetic raw material for the development of the adaptive immune system, and the development of endothelial tissue, greater metabolic activity, and a decreased blood volume-to-body weight ratio, all of which are more specialized in vertebrates than invertebrates and facilitate adaptive immune responses.^[18]

See also

• Omenn syndrome
• Severe combined immunodeficiency

References

1. Jones JM, Gellert M (Aug 2004). "The taming of a transposon: V(D)J recombination and the immune system". Immunological Reviews. 200: 233–48. doi:10.1111/j.0105-2896.2004.00168.x. PMID 15242409. S2CID 12080467.
2. Notarangelo LD, Kim MS, Walter JE, Lee YN (Mar 2016). "Human RAG mutations: biochemistry and clinical implications". Nature Reviews. Immunology. 16 (4): 234–46. doi:10.1038/nri.2016.28. PMC 5757527. PMID 26996199.
3. Oettinger MA, Schatz DG, Gorka C, Baltimore D (Jun 1990). "RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination". Science. 248 (4962): 1517–23. Bibcode:1990Sci...248.1517O. doi:10.1126/science.2360047. PMID 2360047.
4. Ru H, Chambers MG, Fu TM, Tong AB, Liao M, Wu H (November 2015). "Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures". Cell. 163 (5): 1138–1152. doi:10.1016/j.cell.2015.10.055. PMC 4690471. PMID 26548953.
5. Kapitonov VV, Jurka J (June 2005). "RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons". PLOS Biology. 3 (6): e181. doi:10.1371/journal.pbio.0030181. PMC 1131882. PMID 15898832.
6. Panchin Y, Moroz LL (May 2008). "Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes". Biochemical and Biophysical Research Communications. 369 (3): 818–823. doi:10.1016/j.bbrc.2008.02.097. PMC 2719772. PMID 18313399.
7. Yakovenko I, Agronin J, Smith LC, Oren M (2021). "Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination". Frontiers in Immunology. 12: 709165. doi:10.3389/fimmu.2021.709165. PMC 8355894. PMID 34394111.
8. Kapitonov VV, Koonin EV (2015-04-28). "Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon". Biology Direct. 10 (1): 20. doi:10.1186/s13062-015-0055-8. PMC 4411706. PMID 25928409.
9. Huang S, Tao X, Yuan S, Zhang Y, Li P, Beilinson HA, Zhang Y, Yu W, Pontarotti P, Escriva H, Le Petillon Y, Liu X, Chen S, Schatz DG, Xu A (June 2016). "Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination". Cell. 166 (1): 102–14. doi:10.1016/j.cell.2016.05.032. PMC 5017859. PMID 27293192.
10. Morales Poole JR, Huang SF, Xu A, Bayet J, Pontarotti P (June 2017). "The RAG transposon is active through the deuterostome evolution and domesticated in jawed vertebrates". Immunogenetics. 69 (6): 391–400. bioRxiv 10.1101/100735. doi:10.1007/s00251-017-0979-5. PMID 28451741. S2CID 11192471.
11. Zhang Y, Cheng TC, Huang G, Lu Q, Surleac MD, Mandell JD, Pontarotti P, Petrescu AJ, Xu A, Xiong Y, Schatz DG (May 2019). "Transposon molecular domestication and the evolution of the RAG recombinase". Nature. 569 (7754): 79–84. Bibcode:2019Natur.569...79Z. doi:10.1038/s41586-019-1093-7. PMC 6494689. PMID 30971819.
12. Kasahara M, Suzuki T, Pasquier LD (Feb 2004). "On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates". Trends in Immunology. 25 (2): 105–11. doi:10.1016/j.it.2003.11.005. PMID 15102370.
13. Fugmann SD, Messier C, Novack LA, Cameron RA, Rast JP (Mar 2006). "An ancient evolutionary origin of the Rag1/2 gene locus". Proceedings of the National Academy of Sciences of the United States of America. 103 (10): 3728–33. Bibcode:2006PNAS..103.3728F. doi:10.1073/Pnas.0509720103. PMC 1450146. PMID 16505374.
14. Holland LZ, Albalat R, Azumi K, Benito-Gutiérrez E, Blow MJ, Bronner-Fraser M, et al. (Jul 2008). "The amphioxus genome illuminates vertebrate origins and cephalochordate biology". Genome Research. 18 (7): 1100–11. doi:10.1101/gr.073676.107. PMC 2493399. PMID 18562680.
15. Panchin Y, Moroz LL (May 2008). "Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes". Biochemical and Biophysical Research Communications. 369 (3): 818–23. doi:10.1016/j.bbrc.2008.02.097. PMC 2719772. PMID 18313399.
16. Martin EC, Vicari C, Tsakou-Ngouafo L, Pontarotti P, Petrescu AJ, Schatz DG (2020-05-06). "Identification of RAG-like transposons in protostomes suggests their ancient bilaterian origin". Mobile DNA. 11 (1): 17. doi:10.1186/s13100-020-00214-y. PMC 7204232. PMID 32399063.
17. Kasahara M (Oct 2007). "The 2R hypothesis: an update". Current Opinion in Immunology. Hematopoietic cell death/Immunogenetics/Transplantation. 19 (5): 547–52. doi:10.1016/j.coi.2007.07.009. PMID 17707623.
18. van Niekerk G, Davis T, Engelbrecht AM (2015-09-04). "Was the evolutionary road towards adaptive immunity paved with endothelium?". Biology Direct. 10 (1): 47. doi:10.1186/s13062-015-0079-0. PMC 4560925. PMID 26341882.

Further reading

• Sadofsky MJ (Aug 2004). "Recombination-activating gene proteins: more regulation, please". Immunological Reviews. 200: 83–9. doi:10.1111/j.0105-2896.2004.00164.x. PMID 15242398. S2CID 23905210.
• De P, Rodgers KK (Aug 2004). "Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1". Immunological Reviews. 200: 70–82. doi:10.1111/j.0105-2896.2004.00154.x. PMID 15242397. S2CID 22044642.
• Kapitonov VV, Jurka J (Jun 2005). "RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons". PLOS Biology. 3 (6): e181. doi:10.1371/journal.pbio.0030181. PMC 1131882. PMID 15898832.

External links

• Travis J (November 1998). "The Accidental Immune System; Long ago, a wandering piece of DNA—perhaps from a microbe—created a key strategy" (PDF). Science News. 154 (19): 302–303. doi:10.2307/4010948. JSTOR 4010948. A simple explanation of recombination activating gene for the general reader.