Journal: Human Genetics

Article Title: Allele-specific recognition of the 3′ splice site of INS intron 1

doi: 10.1007/s00439-010-0860-1

Figure Lengend Snippet: IVS1-6T relieves the requirement of intron 1 splicing for U2AF35. a Western blot analysis of HeLa cells transfected with the U2AF35ab siRNA (final concentration of 3, 9, 27 and 81 nM) sc scrambled control (68% GC). Antibodies are shown to the right. b RT-PCR with total RNA extracted from HeLa cells that were transfected with INS reporters shown at the bottom. The wild-type reporter D–F contained haplotype IC. RNA products are to the right. Sequence chromatogram shows activation of cr3′ss+81. c Splicing pattern of two-exon minigenes in U2AF35-depleted cells. Final concentration of U2AF35ab was 30 nM. Reporters are shown at the bottom; location of cloning primers is shown in Fig. a. d Inhibition of 3′ss+81 upon cotransfection of U2AF35ab (30 nM) and U2AF65 K (45 nM) siRNAs. e INS splicing pattern upon separate knockdown of U2AF35 a and U2AF35 b isoforms. The final concentration of siRNAs in each lane was 150 nM (100 nM of U2AF35 a and 50 nM of U2AF35 b in lane 3). f The relative expression of U2AF35a and b isoforms in HeLa cells transfected with siRNAs shown at the top . RT-PCR products were digested with Hinf I, which cuts only U2AF35 b (Pacheco et al. ). The last two lanes are Hinf I-digested PCR products amplified using template plasmids carrying U2AF35 a - and - b isoforms as controls. g upper panel. INS splicing pattern in cells with varying U2AF35/U2AF65 ratios. The amount of wild-type plasmid DNAs (shown at the top) was 0.3, 1.0 and 3.0 μg. siRNAs (shown at the bottom) were added at final concentrations of 90 nM (U2AF65 K) and 10 nM (U2AF35ab). Lower panel shows Western blot analysis with U2AF65 and U2AF35 antibodies. Ex, exogenously expressed subunit. h upper panel. Multiple alignment of C3H ZF proteins. Histidines and cysteines (numbered in U2AF35) coordinated to zinc are highlighted in black, residues involved in base stacking interactions in TIS11d in light grey and other TIS11d residues interacting with RNA in dark grey. Lower panel, rescue of INS splicing with wild-type and mutated U2AF35 isoforms. The extent of rescue is shown on a semiquantitave scale, reflecting decrease of isoform 6 and cr3′ss+81 upon cotransfection of U2AF35-depleted cells with the IC D–F reporters. Rescue plasmids are shown at the top

Article Snippet: The membranes were incubated with mouse PTB (kindly provided by Smith), mouse Puf60 (generously provided by Krainer), mouse U2AF65 (Sigma), rabbit U2AF35 (Protein Tech Group), mouse tubulin (ABcam) and rabbit actin (ABcam) antibodies and appropriate horse radish peroxidase-conjugated secondary antibodies (ABcam).

Techniques: Western Blot, Transfection, Concentration Assay, Control, Reverse Transcription Polymerase Chain Reaction, Sequencing, Activation Assay, Cloning, Inhibition, Cotransfection, Knockdown, Expressing, Amplification, Plasmid Preparation

Journal: Human Genetics

Article Title: Allele-specific recognition of the 3′ splice site of INS intron 1

doi: 10.1007/s00439-010-0860-1

Figure Lengend Snippet: Splicing of INS reporters mutated at first exon position of authentic and cryptic 3′ss of intron 1 upon U2AF35 depletion. a Authentic 3′ss. b Cr3′ss+81. The final concentration of the U2AF35ab duplex was 0, 3, 9 and 27 nM. RNA products are shown to the right. The 3′ss consensus of the wild-type reporters was CAG/A ( a ) and CAG/C ( b ); their predicted intrinsic strength is shown in Supplemental Table 2. c – g Consensus sequences for the first 8 positions of human exons. c 30 clones obtained after 6 rounds of selection with U2AF35 (Wu et al. ). d 444 non-coding human exons (Zhang and Chasin ). e 78 disease-causing human pseudoexons that were generated by intronic mutations, most of them in transposable elements (Vorechovsky ). f 25,000 randomly selected human–mouse conserved exons (Carmel et al. ). g 39,862 human exons from the Alternative splicing Database (Stamm et al. ). The relative nucleotide frequencies at each position were plotted with a pictogram utility available at http://genes.mit.edu/pictogram.html . The height of each letter is proportional to the frequency of the corresponding base at the given position

Article Snippet: The membranes were incubated with mouse PTB (kindly provided by Smith), mouse Puf60 (generously provided by Krainer), mouse U2AF65 (Sigma), rabbit U2AF35 (Protein Tech Group), mouse tubulin (ABcam) and rabbit actin (ABcam) antibodies and appropriate horse radish peroxidase-conjugated secondary antibodies (ABcam).

Techniques: Concentration Assay, Clone Assay, Selection, Generated, Alternative Splicing