Scatterplots of the Logarithm Intensities of Splicing-Related Probes

<p>Points are color-coded as indicated.</p

Scatterplots of Normalized Ratios of Splicing-Related Probes

<p>Points are color-coded by their mutant identity. Gray horizontal and vertical reference lines indicate zero expression ratios.</p

Boxplots of Normalized Ratios of Splicing-Related Probes Stratified by Mutants

<p>Splice-junction and exon probe ratios show a shift from the horizontal zero line in the negative direction, whereas intron probe ratios are centered at zero.</p

Graphical Representation of Designs

<div><p>(A) In this representation, vertices correspond to target mRNA samples and edges to hybridizations between two samples. By convention, we place the green-labeled sample at the tail and the red-labeled sample at the head of the arrow.</p><p>(B) Nested design of the experiment. The effect <i>A</i> is nested in <i>S,</i> and <i>S</i> is in turn nested in <i>V</i>. Note that there are two samples <i>(S)</i> for each mutant, but only one sample for the wild-type.</p></div

Venn Diagram of DE Genes from Different Mutants

<p>(A) compares DE genes among the three <i>spt5</i> mutants <i>(spt5–194, spt5–4,</i> and <i>spt5–242).</i> Statistical test shows that the common 43 genes are highly significant, with a <i>p</i>-value <i><</i> 0.001. In (B), <i>spt5</i> refers to the 43 common genes among all <i>spt5</i> mutants. The overlaps between <i>spt5</i> and <i>ceg1–250</i> (40, <i>p <</i> 0.001), <i>spt5,</i> and <i>spt4</i> (8, <i>p<</i> 0.001), <i>spt4</i>, <i>spt5,</i> and <i>ceg1–250</i> (7, <i>p<</i> 0.001) are all significant.</p

<em>Rbfox1</em> Downregulation and Altered <em>Calpain 3</em> Splicing by <em>FRG1</em> in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

<div><p>Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (<em>FRG1</em>) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. <em>FRG1</em> has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in <em>FRG1</em> mice. We find that splicing perturbations parallel the responses of different muscles to <em>FRG1</em> over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of <em>FRG1</em>-affected splicing events. <em>Rbfox1</em> knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the <em>Rbfox1</em> RNA and decreases its stability. Consistent with this, <em>Rbfox1</em> expression is down-regulated in mice and cells over-expressing <em>FRG1</em> as well as in FSHD patients. Among the genes affected is <em>Calpain 3</em>, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In <em>FRG1</em> mice and FSHD patients, the <em>Calpain 3</em> isoform lacking exon 6 (<em>Capn3 E6–</em>) is increased. Finally, <em>Rbfox1</em> knockdown and over-expression of <em>Capn3 E6-</em> inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of <em>FRG1</em>, reducing <em>Rbfox1</em> levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.</p> </div

Alternative splicing changes are a primary consequence of <i>FRG1</i> overexpression.

<p>(a) Examples of alternative exons (<i>Atl2</i>) or introns (<i>Ttn</i>) spliced normally in the mouse model of Duchenne muscular dystrophy, <i>mdx</i> mice, and example of an alternative exon similarly altered in <i>FRG1</i> and <i>mdx</i> mice (<i>Ktn1</i>). (b) RT-PCR analysis of mRNA splicing variants from proliferating (MB) and differentiating (MT) C2C12 muscle cells over-expressing FRG1. Examples of alternative splicing changes present in both MB and MT (<i>Capn3</i>), only in MB (<i>Ablim1</i>), or only in MT (<i>Nasp</i>). Numbers are the percentage of exon inclusion. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons and double lines represent the affected intron.</p

Alternative splicing isoform of <i>Calpain 3</i> increased in <i>FRG1</i> mice and in FSHD patients.

<p>(a) Real-time RT-PCR and immunoblotting analysis confirming that the <i>Capn3</i> alternative splicing isoform lacking exon 6 (<i>Capn3 E6-</i>) is increased in the <i>vastus lateralis</i> muscle from <i>FRG1</i> mice. (b) RT-PCR analysis of <i>CAPN3</i> splicing in human muscle cells derived from three different healthy subjects and three different FSHD patients indicates increased expression of <i>CAPN3 E6-</i> isoform in FSHD patients. Numbers below the image are the percentage of exon skipping. Black boxes illustrate constitutive exons, white boxes alternatively spliced exon. RT-PCR products were quantified using the Typhoon. (c) <i>RBFOX1</i> expression analysis was performed on RNA extracted from the same samples as in (b) by real-time RT-PCR.</p

<i>Rbfox1</i> down-regulation is responsible for significant portion of the splicing alterations in <i>FRG1</i> mice.

<p>(a) Specific <i>Rbfox1</i> knockdown was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing a control non-silencing shRNA or an shRNA specific for <i>Rbfox1</i> (shRNA#1). (b) Examples of alternative splicing changes caused by <i>Rbfox1</i> knockdown are showed. Numbers are the percentage of exon inclusion. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (c) <i>Rbfox1</i> overexpression causes alternative splicing changes opposite to <i>FRG1</i> over-expression. Specific <i>Rbfox1</i> over-expression was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing an empty vector (<i>EV</i>) or a Myc-tagged Rbfox1 (<i>F1</i>) either in proliferating or differentiating C2C12 muscle cells. (d) Examples of alternative splicing changes caused by <i>Rbfox1</i> over-expression are showed. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (e) Selective <i>in vivo</i> association of Rbfox1 to target regions displaying putative Fox binding sites (FBS). RIP experiment on samples from (c) using anti-Myc or control IgG antibodies. Immunoprecipitated material was analyzed by RT-PCR, quantified using the Typhoon, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).</p

RNA map of Rbfox motifs downstream of exons.

<p>Each point represents the average frequency of UGCAUG in the 150 bp downstream of the 260 exons whose inclusion increases (blue triangles), the 273 exons whose inclusion decreases (red squares) or the 821 expressed alternative cassette exons whose splicing did not change in the comparison (gray circles, <i>q</i>>0.2 and |Sepscore|<0.3). Error bars indicate 95% confidence intervals of the mean frequency distribution for this population of background exons.</p