Fig 2: RAG1/2 is required for DUX4/IGH-driven alternative splicing. (A) The putative RSS-like sequences observed in the proximity of double tandem DRE-DRE sites. The ERG, C6orf89, and CLEC12A RSS-like sequences (i.e., RAG1/2-binding site) are highlighted in green. The conserved positions in the heptamer (CACAGTG) and nonamer (ACAAAAACC) are colored in red. The adjacent DRE-DRE sites are shown in yellow. (B) In vitro RAG1/2 cleavage assay. The putative RSSs in ERG, C6orf89, and CLEC12A were subjected to RAG1/2 cleavage. The classical 12/23-RSS substrates were used as positive control. (C) RAG1/2 cleavage efficiency (red) and the number of conservative bases in various RSS sites (blue). (D) The knock-down efficiency of RAG1 and RAG2 shRNAs. The scramble shRNA was used as control. (E-I) RAG1/2 shRNA knock-down assays. The Reh cells that expressed DUX4/IGH were subjected to the RAG1/2 shRNA knock-down assay. The ERGalt biogenesis was monitored by Western blotting (E, F) and the quantitative real-time PCR (G), and production of CLEC12Aalt and C6orf89alt was also monitored by the quantitative real-time PCR (H, I), *, P < 0.05. **, P < 0.01. ***, P < 0.001. Abbreviations: RAG1/2: Recombination-activating genes 1/2; DUX4/IGH: Double homeobox 4 fused with immunoglobulin heavy chain; RSS: Recombination signal sequences; ERG: E-26 transformation-specific (ETS) family related gene; C6orf89: Chromosome 6 open reading frame 89; CLEC12A: C-type lectin domain family 12, member A; shRNA: Short hairpin RNA; ERGalt: E-26 transformation-specific family related gene abnormal transcript; CLEC12Aalt: C-type lectin domain family 12; member A abnormal transcript; C6orf89alt: Chromosome 6 open reading frame 89 abnormal transcript; PCR: Polymerase chain reaction