Journal: Science Advances

Article Title: Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity

doi: 10.1126/sciadv.abn9458

Figure Lengend Snippet: ( A ) Schematic diagrams of minigene constructs used to assess the effects of RNA secondary structure on trans-splicing of sDscam β 4 . ( B ) Predicted competing RNA pairings. Mutations introduced into dsRNA are indicated on the upper or lower mutated sequences (M2, M3, M4). Stem I has been validated in . Green arrows depict the activation of trans-splicing between different sDscam βs. ( C ) Validation of the effects of RNA pairings on trans-splicing by disruptive single mutations (M2, M3, M4) and compensatory double mutations (M24: M2 + M4; M34: M3 + M4). Data are expressed as means ± SD from three independent experiments. ( D ) Validation of trans-splicing at the protein level. The CDS of EGFP was split into two halves (EG and FP), followed by intronic sequences of sDscam β. EG: exon EG with 1 to 154 nt of intron 4 of the β1V13 cassette; FP: exon FG with 272 to 892 nt of intron 4 of β2. ( E ) Fluorescent photos of S2 cells transfected with WT and mutant plasmids containing CDS of EG/FP fused with intronic sequences from the β1V13 cassette and β2 (scale bars, 100 μm). ( F ) Detection of trans-spliced products by Western blotting using anti-EGFP antibody.

Article Snippet: Western blotting primary antibodies were used for relative quantification of EGFP: anti-EGFP tag mouse monoclonal antibody (1:5000; EarthOx, catalog no. E022030-01) and anti–β-actin mouse monoclonal antibody (1:5000; Abcam, catalog no. ab8224, RRID:AB_449644).

Techniques: Construct, Activation Assay, Transfection, Mutagenesis, Western Blot