8 research outputs found
Splicing factors binding sites in the <i>PAH</i> intronic region surrounding the c.1199+17G>A and c.1199+20G>C variants, predicted with ESEFinder (http://rulai.cshl.edu) and HSF (http://www.umd.be/HSF3/) programs.
<p>Effects of variants and deletions introduced in minigenes. WT; wild type.</p
Effect of the modification of the intronic cryptic splice site on minigene splicing profile.
<p>The upper panel shows the location and predicted splice scores of the natural and cryptic (wild type and with the different mutations) splice sites. The intronic cryptic splice site was either abolished by elimination of the GT (c.1199+18G>C mutation) or optimized (c.1199+15A>C/+20G>A mutations). The gels show the RT-PCR results after transfection of the wild-type and modified pSPL3 (A) or pcDNA3.1 (B) minigenes. On the right of the gel is the schematic drawing showing the identity of the bands. HSF: Human Splice Finder (<a href="http://www.umd.be/HSF3/HSF.html" target="_blank">http://www.umd.be/HSF3/HSF.html</a>); MAXENT: MaxEntScan (<a href="http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html" target="_blank">http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html</a>); BDGP: Berkeley Drosophila Genome Project (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>). The estimated percentage of exon inclusion and the cryptic splice site usage (number of clones in which splicing occurred at the +18 splice site out of total analysed, after subcloning and sequencing the exon inclusion amplified product) are shown below each lane.</p
Effect of the optimization of the 5’ splice site of exon 11 on minigenes splicing profile.
<p>The splicing score of exon 11 5’ splice site was optimized in the 3A6T minigene by introducing the c.1199+3G>A and c.1199+6A>T changes as shown in the above panel, along with the predicted scores calculated with HSF (<a href="http://www.umd.be/HSF3/" target="_blank">http://www.umd.be/HSF3/</a>), MaxEntScan (<a href="http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html" target="_blank">http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html</a>) and BDGP (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>). The gel shows the RT-PCR results after transfection of the wild type (wt) and mutant pcDNA3.1 minigenes with and without the optimized 5’ splice site. The estimated percentage of exon inclusion is shown below each lane. On the right of the gel is the schematic drawing showing the identity of the bands.</p
RNA oligonucleotide affinity studies.
<p>A) Schematic representation of the exon 11-intron 11 junction, the predicted binding sites for splicing factors and the RNA oligonucleotides used; B) Western blot gels after pull-down experiments; the blots shown are representative results from three independent pull-down experiments; C) Coomassie stained gels; 15 ÎĽg of HeLa nuclear extract (NE input), corresponding to 1/50 of the total nuclear extract used as input per pull-down reaction, equal amounts of nuclear extract collected after the binding reaction (NE output), and 7.5 ÎĽl (1/6) of the eluates were loaded and separated on an SDS-PAGE gel, and stained with Coomassie; D) Quantification of the pull down experiments: the intensity of the signal from western blots was quantified and normalized to the signal obtained from the pull-down reaction with the WT sequence. Student t-test was used to evaluate the differences, * p<0.05. BL and NE indicate control lanes without RNA oligonucleotides or with nuclear extract alone, respectively.</p
Co-transfection of wild-type and mutant minigenes with adapted U1snRNA constructs.
<p>Different modified U1 snRNA constructs were generated hybridizing to the 5’ splice site of <i>PAH</i> exon 11 (U1 WT), to the intronic cryptic splice site (U1 18GT), or to the intronic cryptic splice site with the mutations +17 (U1 +17) or +20 (U1+20), as shown in the upper panel (A). Panel B shows the results of co-transfecting the different U1 constructs in the wild type (wt) and mutant pSPL3 minigenes and panel C the results obtained with the minigenes carrying the intronic deletions c.1199+13del7, c.1199+17del6 and c.1199+20del5. On the right of the gel is the schematic drawing showing the identity of the bands. In panel B and C the estimated percentage of exon inclusion is shown below each lane.</p
Effect of intronic deletions on minigenes splicing profile.
<p>Deletions c.1199+13del7, c.1199+17del6 and c.1199+20del5, shown in the scheme above, were introduced in the pSPL3 (A) or pcDNA3.1 (B) wild-type minigenes and the effect on splicing examined after transfection in Hep3B cells. The estimated percentage of exon inclusion is shown below each lane. On the right of each gel is the schematic drawing showing the identity of the bands. V, vector sequences.</p
Minigene analysis of the c.1199+17G>C and c.1199+20G>C variants.
<p>Panel A shows the schematics of the pPSL3 construct and the results after transfection in Hep3B cells of wild-type (wt) and mutant minigenes. Panel B shows the schematics of the pcDNA3.1 construct and the results in Hep3B cells. The splice scores according to MaxEnt program (<a href="http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html" target="_blank">http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html</a>) are indicated for each splice site. On the right of each gel is the schematic drawing showing the identity of the bands confirmed by sequencing analysis The estimated percentage of exon inclusion is shown below each lane. V, vector sequences.</p
Effect of distance of the intronic regulatory region to the natural 5’ splice site.
<p>The upper panel shows the location and sequence of the spacer introduced in the pcDNA3.1 wild type minigene between the natural and the cryptic splice sites (GT underlined). The gel shows the results after transfection of the wild-type (WT) minigene and the constructs with 1, 3 or 6 spacers in Hep3B cells. On the right of the gel is the schematic drawing showing the identity of the bands verified by sequence analysis, that showed usage of both the natural and the cryptic splice site (at +24 with 1 spacer, at +36 with 3 spacers and at +54 with 6 spacers). The estimated percentage of exon inclusion is shown below each lane.</p