Carmona LM et al. (2016), Collaboration of RAG2 with RAG1-like proteins d...

ECB-ART-44615

Genes Dev 2016 Apr 15;308:909-17. doi: 10.1101/gad.278432.116.

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Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination.

Carmona LM , Fugmann SD , Schatz DG .

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The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate V(D)J recombination, the process that assembles the B- and T-lymphocyte antigen receptor genes of jawed vertebrates. RAG1 and RAG2 are thought to have arisen from a transposable element, but the origins of this element are not understood. We show that two ancestral RAG1 proteins, Transib transposase and purple sea urchin RAG1-like, have a latent ability to initiate V(D)J recombination when coexpressed with RAG2 and that in vitro transposition by Transib transposase is stimulated by RAG2. Conversely, we report low levels of V(D)J recombination by RAG1 in the absence of RAG2. Recombination by RAG1 alone differs from canonical V(D)J recombination in having lost the requirement for asymmetric DNA substrates, implicating RAG2 in the origins of the "12/23 rule," a fundamental regulatory feature of the reaction. We propose that evolution of RAG1/RAG2 began with a Transib transposon whose intrinsic recombination activity was enhanced by capture of an ancestral RAG2, allowing for the development of adaptive immunity.

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Genes referenced: LOC100887844 LOC115925255

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	Figure 1. V(D)J recombination mediated by RAG1 in the absence of RAG2. (A) Schematic of the ZGR recombination substrate. The GPT gene is inverted by recombination between the Vκ 12RSS (white) and one of the Jκ 23RSSs (black), yielding a signal joint (SJ) and coding joint (CJ) and allowing for GPT expression from the 5′ LTR and MPA resistance. (hygro) Hygromycin resistance gene; (small black arrows) coding joint and signal joint PCR primer-binding sites. (B,C) Quantitation of recombination in 3T3 fibroblast lines. Recombination efficiency for cells expressing the indicated protein or proteins plotted as the number of MPA-resistant colonies per million cells plated, with each dot indicating an independent experiment and the bar height indicating the mean. (Mock) Empty vector control; (c) core RAG proteins; (FL) full-length RAG proteins; (R1cD) D708A active site mutant of the RAG1 core; (R1flD) D708A active site mutant of RAG1 full length. (B) Wild-type 3T3 line. (C) RAG2−/− 3T3 line. (D) Substrate recombination in v-abl cells. (Top) Schematic of a simplified 12/23 inversional substrate. (Bottom) PCR assays for coding or signal joints in genomic DNA from RAG1−/− or RAG2−/− v-abl cells incubated with STI-571 and expressing RAG1 (in RAG2−/−) or RAG2 (in RAG1−/−) from retroviral vectors, representative of at least three experiments. (Plasmid) Reaction spiked with positive control template DNA. (E) Igκ locus recombination in v-abl cells. (Top) Schematic of the Igκ locus. (Bottom) Representative PCR assays for V-to-Jκ1 and V-to-Jκ2 recombination in RAG1−/− or RAG2−/− cells (expressing retroviral RAG2 or RAG1, respectively) or wild-type cells incubated with ST1–571. (Lane 4) No template control. Expected product sizes for V to Jκ1 or V to Jκ2 are indicated.
	Figure 2. RAG1 does not obey the 12/23 rule. (A) Simplified schematic of recombination substrates. The GPT gene is flanked by a pair of 12RSSs (white; 12_12 substrate) or one 12RSS and one 23RSS (black; 12_23 substrate). (B–D) Quantitation of recombination in RAG2−/− 3T3 fibroblast lines. Recombination efficiency is plotted as in Figure 1B. (B) Note the Y-axis scaling. (Left) Linear. (Right) Logarithmic. (C,D) Note the different Y-axis scales for the two plots and that, in C, one data point with a value of 70 was outside the range of the Y-axis, as indicated. (c) Core RAG proteins; (FL) full-length RAG proteins. One tailed t-test; P-values are noted. (ns) Not significant.
	Figure 3. Recombination mediated by HzTransib and purple sea urchin RAG1-like. (A,B) Quantitation of recombination in RAG1−/− 3T3 fibroblast lines. Recombination efficiency is plotted as in Figure 1B. Note that levels of recombination by RAG1 alone in this RAG1−/− line were higher than in wild-type and RAG2−/− lines. (Mock) Empty vector control; (HZ) Hztransib; (SpR1L) spRAG1L; (HZm) active site mutant of Hztransib; (SpR1Lm) active site mutants of spRAG1L. (C) Representative PCR assays for coding or signal joints in genomic DNA from MPA-resistant cells expressing the indicated proteins. Each lane is an independent clone. (Plasmid) Reaction spiked with positive control template DNA; (HZ) Hztransib; (SpR1L) spRAG1L. (D) GST pull-down to detect interactions between the indicated MBP- or GST-tagged proteins. (R1c) RAG1 core; (Hz) HZTransib; (mR2) mouse RAG2; (sR2) shark RAG2; (+) GST alone. (Top) Glutathione pull-down. (Middle and bottom) Starting material lysate. Arrows indicate the expected positions of MBP-RAG1 core and MBP-Hztransib proteins.
	Figure 4. Transib transposition is enhanced by RAG2. (A) Schematic of in vitro transposition assay. Transposition of a donor DNA fragment, a tetracycline resistance gene (TetR) flanked by pair of TIRs or RSSs (black triangles), into target plasmid with the kanamycin resistance gene (KanR) generates double resistance plasmids. (B,D–F) Quantitation of transposition activity as measured after bacterial transformation. The transposition efficiency for the indicated protein or proteins is plotted as the number of transposition events (Kan/Tet double-resistant colonies) normalized to the total amount of plasmid purified from each reaction (Kan-resistant colonies) and multiplied by 106, with each dot representing an independent experiment and the bar height indicating the mean. (Ctr.) No protein; (HZ) Hztransib; (HZm) Hztransib active site mutant; (R2c) RAG2 core. (B) TIR donor; two-tailed t-test. (D,E) RSS donors. (sN) Scrambled nonamer; (sH) scrambled heptamer. In mutant RSS substrates, the heptamer or nonamer was scrambled in one of the two RSSs, with the other RSS remaining intact. (C) Alignment of Hztransib TIRs to consensus 12RSS and 23RSS. (Boxed) Heptamer; (underlined) nonamer; (highlighted) conserved positions of the heptamer crucial for activity; (*) positions conserved between the RSS and Transib TIR. (G) Model for the origin of the RAG proteins and V(D)J recombination. We propose a two-step model in which a Transib element acquired an ancestral RAG2 gene, leading to a “RAG transposon” containing both RAG1 and RAG2, whose disassembled components subsequently gave rise to split antigen receptor genes and the RAG locus of jawed vertebrates, as proposed by Thompson (1995).

References [+] :

Agrawal, Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. 1998, Pubmed