Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript

<div><p>Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the <i>cis</i>-acting elements and <i>trans</i>-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (<i>FGG</i>) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on <i>FGG</i> pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other <i>trans</i>-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for <i>FGG</i> pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating <i>FGG</i> pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.</p> </div

Functional dissection of G-run elements within the pseudoexon sequence.

<p>(top) The complete 75-bp-long pseudoexon sequence and flanking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the star indicates the IVS6-320A>T mutation; the deleted sequences (shaded in gray) are indicated. (bottom) Histograms representing the relative amount of transcripts including or skipping the pseudoexon, calculated for each deletion mutant as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059333#pone-0059333-g002" target="_blank">Figure 2B</a>. Bars represent mean ± SD of 3 independent experiments, each performed in triplicate. The results were analyzed by unpaired t-test. Statistical significance was calculated referring to the M construct (*P<0.05; **P<0.01; ***P<0.001).</p

Effect of hnRNP H and F modulation on the regulation of <i>FGG</i> pseudoexon splicing.

<p>(A) Knockdown of hnRNP H and F. Western blot (left) and corresponding densitometric analysis (middle) demonstrating the actual silencing of hnRNP H and F proteins in RNAi experiments. (right) Relative expression levels of wild-type and pseudoexon-containing transcripts by qRT-PCR. The ratio between the two isoforms in samples silenced for either hnRNP F or H was also calculated. (B) Transient overexpression of hnRNP F. (left) GeneMapper windows displaying fluorescence peaks corresponding to RT-PCR products obtained from the cDNA of cells transfected with constructs expressing the M minigene with or without hnRNP F overexpression. The fluorescence peak areas were measured by the GeneMapper v4.0 software. The X-axis represents data points (size standard peaks are also indicated) and the Y-axis represents fluorescence units. (right) Histograms represent the relative amount of transcripts including or skipping the pseudoexon, as assessed by calculating the ratio of the corresponding fluorescence peak areas (setting the sum of all peaks as 100%). Bars represent mean ± SD of 3 independent experiments, each performed in triplicate. The results were analyzed by unpaired t-test (*P<0.05; **P<0.01; ***P<0.001).</p

Schematic representation of the 75-bp <i>FGG</i> pseudoexon activated by the IVS6-320A>T mutation.

<p>(top) The fibrinogen cluster; boxes and lines represent exons and intronic/intergenic regions, respectively (only exons are drawn to scale); the two parallel slanted lines indicate breaks in the scale. (middle) The <i>FGG</i> minigene (M) cloned in pTargeT vector; the star marks the IVS6-320A>T mutation. (bottom) The complete 75-bp-long pseudoexon sequence and flaking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the strength of pseudoexon splice sites, calculated by using the NNSPLICE 0.9 (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>) and the Netgene2 (<a href="http://www.cbs.dtu.dk/services/NetGene2/" target="_blank">http://www.cbs.dtu.dk/services/NetGene2/</a>) software is reported below the corresponding sequence; G-stretches are shaded in gray.</p