RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination
RAG1 and RAG2 form a tetramer nuclease to initiate V(D)J recombination in developing T and B lymphocytes. The RAG1 protein evolves from a transposon ancestor and possesses nuclease activity that requires interaction with RAG2. Here, we show that the human RAG1 protein aggregates in the nucleus in the absence of RAG2, exhibiting a extremely low V(D)J recombination activity. In contrast, RAG2 does not aggregate by itself, but it interacts with RAG1 to disrupt RAG1 aggregates and thereby to activate robust V(D)J recombination. Moreover, RAG2 from mouse and zebrafish could not disrupt the aggregation of human RAG1 as efficiently as human RAG2 did, indicating a species-specific regulatory mechanism for RAG1 by RAG2. Therefore, we propose that RAG2 coevolves with RAG1 to release inert RAG1 from aggregates and thereby activate V(D)J recombination to generate diverse antigen receptors in lymphocytes. PEM-seq sequencing was used for detecting the catalytic activity of human RAG complex . The Sample name and Species ,cell lines and treatment were listed at a Excell table. Qrignal sequencing and processed data were loaded at the datafiles. The processed data was used the superQ scripts as the descripted (Yin et al., 2019; Liu et al., 2021).
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Low-frequency coding joins generated by RAG recombinase could be hardly distinguish by PCR. Therefore, we used the PEM-seq method to monitor RAG1or RAG complex-generated breaks at RSS sites and a bait site cleaved by RAG complex at a bona fide 23 RSS were used. Translocation junctions between the 23 RSS cutting site and other break sites were retrieved. The plasmids were transfected into 293T-CJ cells with the Ca3(PO4)2 coprecipitation approach and the cells were cultured for 4 days. Cells were harvested, washed with PBS, and subjected to genomic DNA extraction with 1:100 Proteinase K digestion. Next, PEM-Seq libraries were generated as previously described (Yin et al., 2019). In brief, primer extension was performed with biotinylated primers. Then enriched biotinylated PCR products were ligated to the bridge adapters, and two subsequent PCR steps were performed to amplify products for sequencing. Sequences were mapped to the hg19 reference via the SuperQ pipeline (Yin et al., 2019). PEM-seq reads were processed as follows. For initial reads preprocessing, both Illumina 2000 adapter sequences and ending low-quality sequences (QC < 30) were trimmed by cutadapt package (http://cutadapt.readthedocs.io/en/stable/); remaining reads shorter than 25 bp were discarded. Then reads were de-multiplexed using fastq-multx (https://github.com/ brwnj/fastq-multx) to distinguish the index. For reads alignment and clustering, we adapted the corresponding pipeline used in SuperQ to perform reads mapping and break site detection. Note that we used the hg19 genome (modified by DEL-CJ segments insertion at chromosome 1) as reference. Uniquely mapped reads were filtered program as LAM-HTGTS (Hu et al., 2016) did but all the duplicates were kept for the following precession. A molecular barcode (RMB: used to distinguish different reads to remove the replicates from PCR amplification) clustering algorithm38 was adapted to our analysis. The deletional coding joins between the adjacent sequences to 12 and 23 RSSs were counted as V(D)J recombination events. Besides, reads without any detected mutations around break point were identified as germline sequences. Taking uncut primer control and sequencing depth into account, the efficiency of V(D)J recombination for different RAG complex was calculated as bellow: V(D)J recombination frequency (%) = Coding joins/total identified fragments %.