Pedro Miura

Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated β-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.

Pharmacological activation of PPARβ/δ stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

Brain-derived neurotrophic factor expression is repressed during myogenic differentiation by miR-206.

J. Neurochem. (2012) 120, 230–238.

The Utrophin A 5′-Untranslated Region Confers Internal Ribosome Entry Site-mediated Translational Control during Regeneration of Skeletal Muscle Fibers

Utrophin up-regulation in muscle fibers of Duchenne muscular dystrophy patients represents a potential therapeutic strategy. It is thus important to delineate the regulatory events presiding over utrophin in muscle in attempts to develop pharmacological interventions aimed at increasing utrophin expression. A number of studies have now shown that under several experimental conditions, the abundance of utrophin is increased without a corresponding elevation in its mRNA. Here, we examine whether utrophin expression is regulated at the translational level in regenerating muscle fibers. Treatment of mouse tibialis anterior muscles with cardiotoxin to induce muscle degeneration/regeneration led to a large (∼14-fold) increase in the levels of utrophin A with a modest change in expression of its transcript (40%). Isolation of the mouse utrophin A 5′-untranslated region (UTR) revealed that it is relatively long with a predicted high degree of secondary structure. In control muscles, the 5′-UTR of utrophin A caused an inhibition upon translation of a reporter protein. Strikingly, this inhibition was removed during regeneration, indicating that expression of utrophin A in regenerating muscles is translationally regulated via its 5′-UTR. Using bicistronic reporter vectors, we observed that this translational effect involves an internal ribosome entry site in the utrophin A 5′-UTR. Thus, internal ribosome entry site-mediated translation of utrophin A can, at least partially, account for the discordant expression of utrophin A protein and transcript in regenerating muscle. These findings provide a novel target for up-regulating levels of utrophin A in Duchenne muscular dystrophy muscle fibers via pharmacological interventions.

CircRNA accumulation in the aging mouse brain.

Circular RNAs (circRNAs) are a newly appreciated class of RNAs expressed across diverse phyla. These enigmatic transcripts are most commonly generated by back-splicing events from exons of protein-coding genes. This results in highly stable RNAs due to the lack of free 5′ and 3′ ends. CircRNAs are enriched in neural tissues, suggesting that they might have neural functions. Here, we sought to determine whether circRNA accumulation occurs during aging in mice. Total RNA-seq profiling of young (1 month old) and aged (22 month old) cortex, hippocampus and heart samples was performed. This led to the confident detection of 6,791 distinct circRNAs across these samples, including 675 novel circRNAs. Analysis uncovered a strong bias for circRNA upregulation during aging in neural tissues. These age-accumulation trends were verified for individual circRNAs by RT-qPCR and Northern analysis. In contrast, comparison of aged versus young hearts failed to reveal a global trend for circRNA upregulation. Age-accumulation of circRNAs in brain tissues was found to be largely independent from linear RNA expression of host genes. These findings suggest that circRNAs might play biological roles relevant to the aging nervous system.

Modulation of utrophin A mRNA stability in fast versus slow muscles via an AU-rich element and calcineurin signaling

We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3′UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3′UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.

IRES-Mediated Translation of Utrophin A Is Enhanced by Glucocorticoid Treatment in Skeletal Muscle Cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6α−methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5′UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5′UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5′UTR. Additional experiments identified a distinct region within the utrophin A 5′UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.

A 1.3 kb promoter fragment confers spatial and temporal expression of utrophin A mRNA in mouse skeletal muscle fibers

Abstract Upregulation of utrophin in muscle is currently being examined as a potential therapy for Duchenne muscular dystrophy patients. In this context, we generated transgenic mice harboring a 1.3kb human utrophin A promoter fragment driving expression of the lacZ gene. Characterization of reporter expression during postnatal muscle development revealed that the levels and localization of β-galactosidase parallel expression of utrophin A transcripts. Moreover, we noted that the utrophin A promoter is more active in slow soleus muscles. Additionally, expression of the reporter gene was regulated during muscle regeneration in a manner similar to utrophin A transcripts. Together, these results show that the utrophin A promoter- lacZ construct mirrors expression of utrophin A mRNAs indicating that this utrophin A promoter fragment confers temporal and spatial patterns of expression in skeletal muscle. This transgenic mouse will be valuable as an in vivo model for developing and testing molecules aimed at increasing utrophin A expression.

Alternative polyadenylation in the nervous system: To what lengths will 3′ UTR extensions take us?

Alternative cleavage and polyadenylation (APA) can diversify coding and non‐coding regions, but has particular impact on increasing 3′ UTR diversity. Through the gain or loss of regulatory elements such as RNA binding protein and microRNA sites, APA can influence transcript stability, localization, and translational efficiency. Strikingly, the central nervous systems of invertebrate and vertebrate species express a broad range of transcript isoforms bearing extended 3′ UTRs. The molecular mechanism that permits proximal 3′ end bypass in neurons is mysterious, and only beginning to be elucidated. This landscape of neural 3′ UTR extensions, many reaching unprecedented lengths, may help service the unique post‐transcriptional regulatory needs of neurons. A combination of approaches, including transcriptome‐wide profiling, genetic screening to identify APA factors, biochemical dissection of alternative 3′ end formation, and manipulation of individual neural APA targets, will be necessary to gain fuller perspectives on the mechanism and biology of neural‐specific 3′ UTR lengthening.