Nonproductive Splicing Prevents Expression of MYH7b Protein in the Mammalian Heart

Background Although the roles of alpha‐myosin heavy chain (&alpha;‐MyHC) and beta‐myosin heavy chain (&beta;‐MyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes non‐productive alternative splicing that prevents protein expression in a tissue‐specific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts. &nbsp; Methods and Results By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the non‐coding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of full‐length protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples. &nbsp; Conclusions The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody cross‐reactivity. More importantly, our data predict that the MYH7b disease‐associated variants may operate through the alternately spliced RNA itself. &nbsp;</p

Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy

In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH7b null mice to determine the potential role of MYH7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.</p

miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

Background Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions. Methods We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3&prime; UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments. Results Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8. Conclusions Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network. </div

Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

BACKGROUND: Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5′ and 3′ splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. METHODOLOGY/PRINCIPAL FINDING: Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3′ splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites

SR proteins bind exon 23 enhancer elements.

<p>³²P labeled D90 RNA was incubated at 30°C for 10 min with purified SR proteins prepared from calf thymus as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Zahler1" target="_blank">[63]</a>. Binding reactions performed in the absence or in the presence of increasing concentrations of cold competitor RNAs (with molar excess of 30, 60, 90 and 120 fold, as indicated by the gradients above the gel lanes) were fractionated on a 4% non-denaturing polyacrylamide gel. The schematic representation of the RNAs is depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. The positions of free RNA and bound complexes are shown. Comparable results were obtained in two independent experiments.</p

Exon 23 contains purine-rich elements that activate splicing of a heterologous substrate containing a weak 3′ splice site.

<p>The sequence of the first 90 nucleotides of dystrophin exon 23 is shown at the top of the figure. Arrows identify the natural mutation (C→T) occurring in the <i>mdx</i> mouse and the mutations that disrupt the purine-rich region R1. The borders of the M1 element are defined by the homology (12 of 12 positions) shared with the human dystrophin exon 23. Different regions of dystrohin exon 23 were inserted in the second exon of Dpy2, an enhancer-dependent mRNA containing a weak pyrimidine stretch <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Dye1" target="_blank">[60]</a>. The structure of the chimeric pre-mRNA substrates subjected to <i>in vitro</i> splicing reactions is shown at the bottom right of the figure. White boxes indicate Dpy2 exons, black boxes indicate different regions of the dystrophin exon 23, dashed boxes indicate portions of exon 23 not included in the substrates, white vertical lines indicate the <i>mdx</i> and R1 mutations. The gray box in Dpy2E identifies the two splicing enhancers found in the α-tropomyosin exon 2 gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-Dye1" target="_blank">[60]</a>. The structures and the mobility of the products and intermediates of splicing are shown on the sides of the gel. D172-212Δ RNA was used as a positive control for D1-40Δ to rule out any exon size inhibitory effect on splicing. Asterisks indicate the position of the spliced RNAs. Splicing reactions, carried out for 70 min at 30°C, were resolved on a 12% denaturing polyacrylamide gel. Comparable results were obtained in three independent experiments.</p

Splicing of dystrophin mini-genes <i>in vivo.</i>

<p>Cos 7 cells were transfected with plasmids expressing different portions of the genomic region of the dystrophin gene spanning from exon 22 to exon 23/24. Northern blot analysis was carried out on total RNA prepared 24 hr after transfection, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. Band intensities, quantified by phosphorimager analysis, were normalized for transfection efficiency and RNA recovery to the level of the co-expressed neomycin mRNA (neo). Normalized values were expressed as a percentage of wt mRNAs that were defined as 100. The schematic structure of the substrates is shown on the right part of both panels A and B. Dystrophin exons are identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone-0000427-g001" target="_blank">Figure 1</a>. β-globin exons are shown as gray boxes while the 5′ and 3′ UTRs as white thinner boxes. (A) Two exon mini-genes carrying dystrophin exons 22 and 23. (B) Three exon mini-genes carrying dystrophin exon 22 and 23 and 24, chimeric constructs, and the β-globin system (β−globin wt and β−globin 39 carrying a nonsense mutation at codon 39) used as a reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#pone.0000427-LykkeAndersen1" target="_blank">[81]</a>. Comparable results were obtained in three independent experiments. (C) Left panel: autoradiogram of a representative quantitative RT-PCR performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. The upper bands correspond to RT-PCR products from dystrophin mRNA obtained with primers located in exons 22 and 24; the lower bands correspond to the neomycin gene used as an internal control. −and+lanes correspond to reactions carried out in absence or presence of reverse transcriptase. Right panel: non-quantitative RT-PCR performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000427#s4" target="_blank">Materials and Methods</a>. Mobility of the predicted splicing products is shown on the right. The PCR product deriving from skipping of exon 23 was not detected.</p