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

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

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

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

<div><p>Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in <i>COL4A3</i>, <i>COL4A4</i>, and <i>COL4A5</i> genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting <i>COL4A5</i> exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel <i>COL4A5</i> hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in <i>COL4A3</i> exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.</p></div

<i>In-vitro</i> analysis of the impact of c.2245-40A>G variant on <i>COL4A5</i> pre-mRNA splicing.

<p>(A) Schematic representation of the hybrid pBS-KS-COL4A5_ex29 minigene where α-globin exons are represented by light grey boxes, fibronectin (<i>FN1</i>) exons by white boxes, whereas introns are shown as black lines (not to scale). Exon 29 of <i>COL4A5</i> is represented by a dark grey box. The c.2245-40A>G mutation in intron 28 is indicated by a star. Primers used in RT-PCR assays are also indicated. (B) On the left, agarose gel (2%) electrophoresis of RT-PCR products obtained from RNA of HEK293 cells transfected with the wild-type (wt) or mutant (mut) minigene vector. M: molecular weight marker (pUC9-<i>Hae</i>III). In the middle, GeneMapper windows show fluorescence peaks corresponding to the molecular species amplified by RT-PCR. Grey shaded peaks correspond to the RT-PCR-labeled products, whose relative quantitation is reported on the right of the panel (%). Unshaded peaks represent the size standard (ROX-500 HD). The <i>x</i> axis indicates fluorescence units. On the right, schematic representation of the splicing products, as verified by Sanger sequencing. The length of each fragment is shown.</p

<i>In-vitro</i> analysis of the impact of c.2245-40A>G variant on <i>COL4A5</i> pre-mRNA splicing.

<p>(A) Schematic representation of the hybrid pBS-KS-COL4A5_ex29 minigene where α-globin exons are represented by light grey boxes, fibronectin (<i>FN1</i>) exons by white boxes, whereas introns are shown as black lines (not to scale). Exon 29 of <i>COL4A5</i> is represented by a dark grey box. The c.2245-40A>G mutation in intron 28 is indicated by a star. Primers used in RT-PCR assays are also indicated. (B) On the left, agarose gel (2%) electrophoresis of RT-PCR products obtained from RNA of HEK293 cells transfected with the wild-type (wt) or mutant (mut) minigene vector. M: molecular weight marker (pUC9-<i>Hae</i>III). In the middle, GeneMapper windows show fluorescence peaks corresponding to the molecular species amplified by RT-PCR. Grey shaded peaks correspond to the RT-PCR-labeled products, whose relative quantitation is reported on the right of the panel (%). Unshaded peaks represent the size standard (ROX-500 HD). The <i>x</i> axis indicates fluorescence units. On the right, schematic representation of the splicing products, as verified by Sanger sequencing. The length of each fragment is shown.</p

Identification of candidate <i>COL4A5</i> and <i>COL4A3</i> variants segregating with Alport syndrome in three Italian families.

<p>Pedigrees of Family 1 (A, X-linked), 2 (B, X-linked), and 3 (C, autosomal recessive) showing the segregation of the identified variants with AS. Individuals analyzed by WES are pointed by an arrow. The genotype of available individuals from each family is indicated below the corresponding symbols and illustrative electropherograms are shown on the right. <u>M</u>, mutant; W, wild type; R, A or G; S, G or C; Y, C or T; CKD, chronic kidney disease.</p

Functional characterization of the signal peptide deletion in COL4A3.

<p>Single confocal sections of HEK293 cells expressing EGFP N-terminus fused either with the entire COL4A3 signal peptide (SP-wt-hybEGFP, top panels) or the 8-amino-acid deleted signal peptide (SP-del-hybEGFP, middle panels). Positive control cells, expressing a soluble EGFP (pEGFP-N1) are also shown (bottom panels). DAPI, 4',6-diamidino-2-phenylindole; EGFP, Enhanced green fluorescent protein. Scale bar: 10 μm.</p

Patient characteristics.

<p>Patient characteristics.</p