SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.

Guanine-quadruplexes (G4) included in RNA molecules exert several functions in controlling gene expression at post-transcriptional level; however, the molecular mechanisms of G4-mediated regulation are still poorly understood. Here, we describe a regulatory circuitry operating in the early phases of murine muscle differentiation in which a long non-coding RNA (SMaRT) base pairs with a G4-containing mRNA (Mlx-γ) and represses its translation by counteracting the activity of the DHX36 RNA helicase. The time-restricted, specific effect of lnc-SMaRT on the translation of Mlx-γ isoform modulates the general subcellular localization of total MLX proteins, impacting on their transcriptional output and promoting proper myogenesis and mature myotube formation. Therefore, the circuitry made of lnc-SMaRT, Mlx-γ, and DHX36 not only plays an important role in the control of myogenesis but also unravels a molecular mechanism where G4 structures and G4 unwinding activities are regulated in living cells.

Detrimental effect of class-selective histone deacetylase inhibitors during tissue regeneration following hindlimb ischemia.

Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreated mice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formed muscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereas MS275 inhibited it. Capillarogenesis did not change. Chromatin immunoprecipitations revealed that class IIa HDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues.

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy.

Duchenne muscular dystrophy (DMD)--which is caused by mutations in the dystrophin gene-is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA--miR-31--that represses dystrophin expression by targeting its 3' untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

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 Δ44 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.

A KO mouse model for the lncRNA Lhx1os produces motor neuron alterations and locomotor impairment.

Here, we describe a conserved motor neuron-specific long non-coding RNA, Lhx1os, whose knockout in mice produces motor impairment and postnatal reduction of mature motor neurons (MNs). The ER stress-response pathway result specifically altered with the downregulation of factors involved in the unfolded protein response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function.

Lnc-SMaRT Translational Regulation of Spire1, A New Player in Muscle Differentiation.

The destiny of a messenger RNA is determined from a combination of in cis elements, like peculiar secondary structures, and in trans modulators, such as RNA binding proteins and non-coding, regulatory RNAs. RNA guanine quadruplexes belong to the first group: these strong secondary structures have been characterized in many mRNAs, and their stabilization or unwinding provides an additional step for the fine tuning of mRNA stability and translation. On the other hand, many cytoplasmic long non-coding RNAs intervene in post-transcriptional regulation, frequently by direct base-pairing with their mRNA targets. We have previously identified the lncRNA SMaRT as a key modulator of the correct timing of murine skeletal muscle differentiation; when expressed, lnc-SMaRT interacts with a G-quadruplex-containing region of Mlx-γ mRNA, therefore inhibiting its translation by counteracting the DHX36 helicase activity. The "smart" mode of action of lnc-SMaRT led us to speculate whether this molecular mechanism could be extended to other targets and conserved in other species. Here, we show that the molecular complex composed by lnc-SMaRT and DHX36 also includes other mRNAs. We prove that lnc-SMaRT is able to repress Spire1 translation through base-pairing with its G-quadruplex-forming sequence, and that Spire1 modulation participates to the regulation of proper skeletal muscle differentiation. Moreover, we demonstrate that the interaction between DHX36 and lnc-SMaRT is indirect and mediated by the mRNAs present in the complex. Finally, we suggest an extendibility of the molecular mechanism of lnc-SMaRT from the mouse model to humans, identifying potential functional analogues.

The Dof protein DAG1 mediates PIL5 activity on seed germination by negatively regulating GA biosynthetic gene AtGA3ox1.

We have previously shown that inactivation of the gene encoding the Arabidopsis thaliana transcription factor DOF AFFECTING GERMINATION 1 (DAG1) renders seed germination more sensitive to both phytochrome B (phyB) and gibberellins (GA). dag1 mutant seeds require less red (R) light fluence and a lower GA concentration than WT to germinate. Here, we show that inactivation of the gene PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) results in down-regulation of DAG1. Inactivation of PIL5 in the dag1 mutant background further increased the germination potential of dag1 mutant seeds, supporting the suggestion that DAG1 is under the positive control of PIL5. Germination of dag1phyB seeds showed a reduced requirement of gibberellins as compared with phyB mutant seeds, both in the presence and in the absence of GA biosynthesis. Furthermore, the GA biosynthetic gene AtGA3ox1 is upregulated in dag1 seeds as compared with the WT, and DAG1 actually binds to the AtGA3ox1 promoter, as shown by chromatin immunoprecipitation experiments. Expression analysis at different time points confirms that AtGA3ox1 is directly regulated by DAG1, while suggesting that DAG1 is not a direct regulatory target of PIL5. Our data indicate that in the phyB pathway leading to seed germination, DAG1 negatively regulates GA biosynthesis and suggest that DAG1 acts downstream of PIL5. In addition, the analysis of hypocotyls of dag1 and phyB mutant plantlets, of plantlets overexpressing phyB in the dag1 mutant, as well as of dag1phyB double mutant suggests that DAG1 may act as a negative regulatory element downstream of phyB also in hypocotyl elongation.

Visualization of Nuclear and Cytoplasmic Long Noncoding RNAs at Single-Cell Level by RNA-FISH.

The RNA fluorescence in situ hybridization (RNA-FISH) methodology offers an attractive strategy to deepen our knowledge on the long noncoding RNA biology. In this chapter, we provide a comprehensive overview of the current RNA-FISH protocols available for imaging nuclear and cytoplasmic lncRNAs within cells or tissues. We describe a multicolor approach optimized for the simultaneous visualization of these transcripts with their specific molecular interactors, such as proteins or DNA sequences. Common challenges faced by this methodology such as cell-type specific permeabilization, target accessibility, image acquisition, and post-acquisition analyses are also discussed.

A longitudinal study defined circulating microRNAs as reliable biomarkers for disease prognosis and progression in ALS human patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis. To date, multiple panels of biomarkers have been described in ALS patients and murine models. Nevertheless, none of them has sufficient specificity and thus the molecular signature for ALS prognosis and progression remains to be elucidated. Here we overcome this limitation through a longitudinal study, analyzing serum levels of circulating miRNAs, stable molecules that are recently used as promising biomarkers for many types of human disorders, in ALS patients during the progression of the pathology. We performed next-generation sequencing (NGS) analysis and absolute RT quantification of serum samples of ALS patients and healthy controls. The expression levels of five selected miRNAs were quantitatively analyzed during disease progression in each patient and we demonstrated that high levels of miR-206, miR-133a and miR-151a-5p can predict a slower clinical decline of patient functionality. In particular, we found that miR-206 and miR-151a-5p serum levels were significantly up-regulated at the mild stage of ALS pathology, to decrease in the following moderate and severe stages, whereas the expression levels of miR-133a and miR-199a-5p remained low throughout the course of the disease, showing a diagnostic significance in moderate and severe stages for miR-133a and in mild and terminal ones for miR-199a-5p. Moreover, we found that miR-423-3p and 151a-5p were significantly downregulated respectively in mild and terminal stages of the disease. These data suggest that these miRNAs represent potential prognostic markers for ALS disease.

Trans-generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy.

Exon skipping is an effective strategy for the treatment of many Duchenne Muscular Dystrophy (DMD) mutations. Natural exon skipping observed in several DMD cases can help in identifying novel therapeutic tools. Here, we show a DMD study case where the lack of a splicing factor (Celf2a), which results in exon skipping and dystrophin rescue, is due to a maternally inherited trans-generational epigenetic silencing. We found that the study case and his mother express a repressive long non-coding RNA, DUXAP8, whose presence correlates with silencing of the Celf2a coding region. We also demonstrate that DUXAP8 expression is lost upon cell reprogramming and that, upon induction of iPSCs into myoblasts, Celf2a expression is recovered leading to the loss of exon skipping and loss of dystrophin synthesis. Finally, CRISPR/Cas9 inactivation of the splicing factor Celf2a was proven to ameliorate the pathological state in other DMD backgrounds establishing Celf2a ablation or inactivation as a novel therapeutic approach for the treatment of Duchenne Muscular Dystrophy.

Non-coding RNAs Shaping Muscle.

In 1957, Francis Crick speculated that RNA, beyond its protein-coding capacity, could have its own function. Decade after decade, this theory was dramatically boosted by the discovery of new classes of non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and circular RNAs (circRNAs), which play a fundamental role in the fine spatio-temporal control of multiple layers of gene expression. Recently, many of these molecules have been identified in a plethora of different tissues, and they have emerged to be more cell-type specific than protein-coding genes. These findings shed light on how ncRNAs are involved in the precise tuning of gene regulatory mechanisms governing tissues homeostasis. In this review, we discuss the recent findings on the mechanisms used by lncRNAs and circRNAs to sustain skeletal and cardiac muscle formation, paying particular attention to the technological developments that, over the last few years, have aided their genome-wide identification and study. Together with lncRNAs and circRNAs, the emerging contribution of Piwi-interacting RNAs and transfer RNA-derived fragments to myogenesis will be also discussed, with a glimpse on the impact of their dysregulation in muscle disorders, such as myopathies, muscle atrophy, and rhabdomyosarcoma degeneration.

The Long Non-coding RNA lnc-31 Interacts with Rock1 mRNA and Mediates Its YB-1-Dependent Translation.

Cytoplasmic long non-coding RNAs have been shown to act at many different levels to control post-transcriptional gene expression, although their role in translational control is poorly understood. Here, we show that lnc-31, a non-coding RNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing its translational activator, YB-1. We find that lnc-31 binds to the Rock1 mRNA as well as to the YB-1 protein and that translational activation requires physical interaction between the two RNA species. These results suggest a localized effect of YB-1 stabilization on the Rock1 mRNA. ROCK1 upregulation by lnc-31, in proliferative conditions, correlates well with the differentiation-repressing activity of ROCK1. We also show that, upon induction of differentiation, the downregulation of lnc-31, in conjunction with miR-152 targeting of Rock1, establishes a regulatory loop that reinforces ROCK1 repression and promotes myogenesis.

U1 snRNA as an effective vector for stable expression of antisense molecules and for the inhibition of the splicing reaction.

We report the use of the U1 snRNA as a vector for the stable expression of antisense molecules against the splice junctions of specific dystrophin exons. The single-stranded 5' terminus of U1 can be replaced by unrelated sequences as long as 50 nucleotides without affecting both the stability and the ability to assemble into snRNP particles. Effective exon skipping has been obtained for different dystrophin exons by antisense sequences against 5' and 3' splice sites alone or in combination with ESE sequences. The efficacy of these molecules has been studied both in in vitro systems and in animals. In both cases the chimeric molecules, delivered as part of lentiviral or AAV vectors (De Angelis et al. Proc Natl Acad Sci USA 99:9456-9461, 2002; Denti et al. Proc Natl Acad Sci USA 103: 3758-3763, 2006; Denti et al. Hum Gene Ther 17: 565-743, 2006; Denti et al. Hum Gene Ther 19: 601-608, 2008; Incitti et al. Mol Ther 18: 1675-1682, 2010), provided high skipping activity and efficient rescue of dystrophin synthesis. Moreover, the U1-antisense molecules, delivered to mice via systemic injection of recombinant AAV viruses, displayed body wide transduction, long-term expression, dystrophin rescue as well as morphological and functional benefit (Denti et al. Hum Gene Ther 19: 601-608, 2008). In this Chapter we report methods for producing U1-antisense expression cassettes in the backbone of lentiviral constructs and for testing their activity both in patients' derived myoblasts as well as in fibroblasts reprogrammed to muscle differentiation.

miRNAs as serum biomarkers for Duchenne muscular dystrophy.

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle-miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild-type levels in animals 'cured' through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR-1, miR-133, and miR-206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

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.