The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype

Substitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes

A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells

Several vectors for the induction of RNA interference in mammalian cells have been described,based mainly on polIII-dependent promoters. They transcribe short hairpin RNAs (shRNA) that,after being processed into short interfering RNAs (siRNAs), mediate the degradation of the target mRNA. Here, we describe the construction of a new siRNA-expressing vector (psiUx) based on the strong and ubiquitous polII-dependent promoter of the human U1 small nuclear RNA (snRNA)gene. In psiUx, the only constraint for the shRNA sequence is a purine at position +1, since specific 3'-end formation is achieved by a box element located downstream of the transcribed region. Several constructs were designed against the lamin A/C target. Depending on the structure of the shRNA transcribed, a preferential or exclusive accumulation of the antisense strand is obtained, thus avoiding possible nonspecific targeting by the sense strand. In all cases tested, very effective siRNAs were produced, thus providing a proof-of-principle that a snRNA-type polII promoter can be used for the expression of siRNAs. We show that psiUx ensures high levels of expression and efficient knock down of the target gene also in stable cell lines

CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery

Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl) ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl) ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl) ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl) ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibitDNMT1activity, whereas poly(ADP-ribosyl) ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl) ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code

Long-term benefit of adeno-associated virus/antisense-mediated exon skipping in dystrophic mice

Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology

Exon 45 Skipping Through U1-snRNA Antisense Molecules Recovers the Dys-nNOS Pathway and Muscle Differentiation in Human DMD Myoblasts

Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Delta 44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway. Received 10 May 2012; accepted 27 July 2012; advance online publication 11 September 2012. doi:10.1038/mt.2012.17

MicroRNAs Involved in Molecular Circuitries Relevant for the Duchenne Muscular Dystrophy Pathogenesis Are Controlled by the Dystrophin/nNOS Pathway

In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program