The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping

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

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

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

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

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