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

Effects of KRT14 depletion on KRT14, E2F1, cyclin E1 and cyclin D1 mRNA levels and cell growth in human pulmonary cell line H441.

<p>A) KRT14 mRNA levels following siRNA-mediated depletion (siKRT14), day two after siKRT14 transfection, n = 6, *p<0.05 compared to siGL2. E2F1, cyclin E1 and cyclin D1 mRNA levels following cell treatment by siKRT14, day two after siKRT14 transfection, n = 6. B) Cell count at day two after KRT14 depletion by siRNA (siKRT14) compared with control siRNA (siGL2). All data are reported as mean ± SD.</p

List of controls and patients screened for Keratin 14 (KRT14) expression.

<p>List of controls and patients screened for Keratin 14 (KRT14) expression.</p

Effects of E2F1 depletion/overexpression in the human pulmonary cell line H441.

<p>A) Cell count at day 0,1,2, and 3 after E2F1 depletion by siRNA (siE2F1) compared with control siRNA (siGL2), n = 4, +p<0,05 compared to siGL2. B) mRNA levels of KRT14, E2F1, cyclin E1 and cyclin D1 at day 2 after cell cycle arrest caused by E2F1 depletion (siE2F1), n = 6, *p<0.05 compared to siGL2. C) Protein levels of KRT14 and E2F1 in H441 assessed by Western blot at day 2 following E2F1 depletion by siE2F1; GAPDH level was used for normalization. D) Cell count at day 0,1,2, and 3 after E2F1 overexpression, n = 3, ^p<0.05 compared to pEGFP. E) mRNA expression levels of KRT14, E2F1, cyclin E1 and cyclin D1 at day 3 after cell cycle progression induced by E2F1 overexpression (pE2F1) compared with an unrelated transfection vector (pEGFP), n = 4, *p<0.05 compared to pEGFP. All data are reported as mean ± SD.</p

Kinetic of KRT14, E2F1, cyclinE1 and cyclin D1 expression in human pulmonary cell lines A549 and H441.

<p>A) KRT14 mRNA levels in A549 and H441 lines at day 1 and 2 under basal growth conditions; n = 6, *p<0,05 compared to day 1, data were normalized to the levels of 28S. B) E2F1 mRNA level in A549 and H441 lines at day 1 and 2 under basal growth conditions, n = 6, § p<0.05 compared to day 2, data were normalized to the levels of 28S. C) Protein level of KRT14 and E2F1 in H441 assessed by Western blot at day 1 and 2 under basal growth conditions; GAPDH level was used for normalization. D) Cyclin E1 mRNA expression level in H441 at day 1 and 2 under basal growth conditions, n = 5, + p<0.001 compared to day 2, data were normalized to the levels of 28S. E) Cyclin D1 mRNA level in H441 at day 1 and 2 under basal growth conditions, n = 5, # p<0.001 compared to day 2, data were normalized to the levels of 28S. All data are reported as mean ± SD.</p

Expression of KRT14 in ARDS, ILD and normal lungs.

<p>A) Assessment of Surfactant-B (SP-B) expression at mRNA level. Box plots of the real-time PCR measurements as ΔCt (SP—B-RLP13a) values of ARDS patients (n = 4). ILD patients (n = 4) and Controls (n = 6). The box whisker plots visualize the minimum (end of the bottom whisker), the first quartile (bottom border of the box), the median (line through the box), the third quartile (top border of the box), and the maximum (end of the top whisker) of the distribution. DAD = diffuse alveolar damage, ILD = interstitial lung disease. B) Assessment of KRT14 expression at mRNA level. Box plots of the real-time PCR measurements as ΔCt (KRT14-RPL13a) values of ARDS patients (n = 4), ILD patients (n = 4) and Controls (n = 6). ΔCt values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172130#pone.0172130.t001" target="_blank">Table 1</a>. The box whisker plots visualize the minimum (end of the bottom whisker), the first quartile (bottom border of the box), the median (line through the box), the third quartile (top border of the box), and the maximum (end of top whisker) of the distribution. C) Assessment of KRT14 expression at protein level. The immunoreactive material with MW compatible with that of KRT14 protein only in DAD patients. Upper panel: Western blot analysis. Lower panel: Ponceau red staining. The loading order is indicated in between the two panels and the Subject numbers correspond to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172130#pone.0172130.t001" target="_blank">Table 1</a>.</p

Kinetic of human pulmonary cell lines A549 and H441 growth.

<p>A) Cell count of A549 and H441 lines at day 0, 1, 2, and 3 in basal growth conditions. B) Cell viability of A549 and H441 lines at day 0, 1, 2, and 3 in basal growth conditions. All data are reported as mean ± SD.</p