The weak 3’ splice site in <i>HRAS</i> exon 2 has a non-consensus G nucleotide and a GGG triplet in the polypyrimidine tract.

<p>(<b>a</b>) Representative results from HepG2 cells transfected with wild type and c.35_36GC>TG mutant minigenes holding either a weak wild type 3’ splice site or an optimized 3’-splice site. Introducing a strong 3’ splices site eliminates skipping of exon 2 indicating that the vulnerability of exon 2 is determined by the weak 3’ splice site. The lane labelled “Vect.” shows the results from a sample transfected with an empty p.cDNA3.1+ vector. (<b>b</b>) Displays the <i>HRAS</i> minigene construct. Sequences of the wild type and optimized 3’-splice sites are displayed. Scores based on MaxEnt calculations for wild type and optimized 3’-splice sites are listed. The mean score for all 3’-splice sites in the <i>HRAS</i> gene is shown. (<b>c</b>) Wild type, c.35_36GC>TG mutant and 6 bp deletion sequences are shown. The scores from ESE-finder and generation of an inhibitory GGG triplet are shown. (<b>d</b>) When a 6 bp deletion is introduced, exon 2 is completely skipped. The lane labelled “Vect.” shows the results from a sample transfected with an empty p.cDNA3.1+ vector.</p

SSO-mediated skipping of <i>HRAS</i> exon 2.

<p>(<b>a</b>) T24 bladder cancer cells, which harbor the c.35G>T mutation, were transfected with <i>HRAS</i> minigenes with either a wild type or an optimized 3’ splice site and treated either with an SSO (SSO-A) that blocks access to the ESE or a scrambled control SSO. SSO-A treatment mediates exon 2 skipping from the wild type <i>HRAS</i> minigene, but this is alleviated when optimizing the 3’ splice site. (<b>b</b>) SSO-A treatment causes nearly complete skipping of endogenous <i>HRAS</i> exon 2 in T24 cells. (<b>c</b>) Western blot analysis confirmed reduced levels of HRAS protein following SSO-A treatment. (<b>d</b>) Quantification of cell viability after SSO-A treatment by WST-1 assay demonstrates that it decreases viability of T24 bladder cancer cells. (<b>e</b>) xCelligence real time monitoring of proliferation of T24 bladder cancer cells. Cells were treated with either SSO-A or control SSO at two concentrations (20 nM or 30 nM). When treated with SSO-A cell viability and growth is decreased.</p

p.Gly12Val mutations in codon 12 of <i>HRAS</i> exon 2 affect splicing differently.

<p>(<b>a</b>) Displays the <i>HRAS</i> minigene construct and the wild type and mutant sequences. The <i>HRAS</i> minigene consisted of the first four <i>HRAS</i> exons (including the natural intronic sequences) cloned into the polylinker of a pcDNA3.1+ vector. (<b>b</b>) Representative results from HepG2 cells transfected with wild type and mutant minigenes. Splicing analysis by PCR amplification and agarose gel electrophoresis reveals extensive exon 2 skipping from c.35_36GC>TG construct and moderate exon 2 skipping from c.35_36GC>TA construct. The lane labelled “Vect.” shows the results from a sample transfected with an empty p.cDNA3.1+ vector. (<b>c</b>) Quantification of the exon 2 inclusion rate from triplicate transfections using a fragment analyzer. Numbers are % inclusion. Calculations are based on molar ratios.</p

Binding analysis of SRSF2 and hnRNPF/H.

<p>(<b>a</b>) Biotinylated RNA oligonucleotides with either wild type or c.35_36TG <i>HRAS</i> sequence were incubated with HeLa nuclear extracts (NE). Biotinylated RNA oligonucleotides bind streptavidin coated beads allowing identification of protein-RNA motif interactions from NE. The beads are superparamagnetic and RNA binding proteins are purified when an external magnetic field is applied. (<b>b</b>) Western blot analysis shows that the c.35_36GC>TG mutation increases binding of hnRNPF/H proteins and decreases binding of SRSF2. (<b>c</b>) siRNA mediated knock down of SRSF2 causes exon 2 skipping both from the wild type <i>HRAS</i> minigene and endogenous <i>HRAS</i> in HepG2 cells. (<b>d</b>) Western blot analysis was used to confirm SRSF2 knock down.</p

Mutations in codon 12 or 13 of <i>HRAS</i> exon 2 affect splicing differently.

<p>(<b>a</b>,<b>b</b>) HepG2 (<i>top</i>) or T24 (<i>bottom</i>) cells were transfected with <i>HRAS</i> minigenes harboring different sequence variants in positions c.34-38. The frequencies of the mutations in Costello syndrome according to Giannoulatou and co-workers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006039#pgen.1006039.ref008" target="_blank">8</a>] and in cancer according to Cosmic database are displayed. For cancer the numbers are displayed with skin cancers included or excluded due to the extremely high occurrence of the c.37G>C mutation in skin cancer. The original scoring of the transforming potential of the mutants in two studies are displayed—A is from Seeburg and co-workers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006039#pgen.1006039.ref005" target="_blank">5</a>]; B is from Fasano and co-workers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006039#pgen.1006039.ref006" target="_blank">6</a>]. Quantitative data for exon 2 inclusion (molar ratio) were obtained from triplicates of duplicate transfections using the Agilent 2100 Bioanalyzer. It is worth noting that there is a clear difference in the overall splicing efficiency between T24 cells and HepG2 cells, which is consistent with the reported low levels of hnRNPF in HepG2 cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006039#pgen.1006039.ref034" target="_blank">34</a>].</p