Journal: PLoS ONE
Article Title: BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development
Figure Lengend Snippet: BCAS2 does not regulate the transcription initiation of Delta but is involved in Delta pre-mRNA splicing. (A) Control of Dl-lacZ (red) in the en > GFP wing disc stained with anti-β-gal antibody. (B) en > GFP , dBCAS2 dsRNA . In the dBCAS2 -depleted posterior compartment, marked by GFP, the expression of Dl-lacZ (red) gives a signal of similar strength as the normal anterior compartment. The expression of GFP and β-galactosidase were merged and displayed in the right panel. Images were taken by confocal microscopy, scale bar, 50 μm. (C). RNA expression of β-galactosidase. RNAs were extracted from wing discs of third instar larvae and subjected to RT-PCR to confirm the RNA expression of β-galactosidase driven by Dl promoter in Act > dBCAS2 dsRNA (lane 2) compared with the control (lane 1). The internal control, rp49 . (D) Schematic diagram of primer design for detecting the intron-containing precursor mRNA (upper) and mRNA of Delta (lower). Primers, exons and introns are denoted with arrowheads, boxes and lines, respectively. (E) Coexpression of dBCAS2 and dBCAS2 dsRNA in larvae can rescue the phenotypes of mRNA decrease and pre-mRNA accumulation caused by dBCAS2 dsRNA . The pre-mRNA and mRNA of Delta were analyzed by quantitative RT-PCR and described in the Materials and Methods. Each genotype was under the control of Act5c-GAL4 driver. White bar: Act5c > +; black bar: Act5c > dBCAS2 dsRNA ; gray bar: Act5c > dBCAS2 dsRNA , dBCAS2 . Data are shown as means and SD relative to the controls from three independent experiments. The P-values was measured by the Student’s t-test. * p
Article Snippet: In vivo splicing assays For the detection of mRNA and pre-mRNA in Drosophila , total RNA from imaginal wing discs in 20 third instar larvae was isolated by using TRI reagent (Sigma-Aldrich).
Techniques: Staining, Expressing, Confocal Microscopy, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Quantitative RT-PCR