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 >2500 fruitfly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and 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 >1000 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 dramatically relative to linear isoforms during CNS aging, and constitute a novel aging biomarker.

Remarkable advances in techniques for gene expression profiling have radically changed our knowledge of the transcriptome. Recently, the mammalian brain was reported to express many long intergenic noncoding (lincRNAs) from loci downstream from protein-coding genes. Our experimental tests failed to validate specific accumulation of lincRNA transcripts, and instead revealed strongly distal 3′ UTRs generated by alternative cleavage and polyadenylation (APA). With this perspective in mind, we analyzed deep mammalian RNA-seq data using conservative criteria, and identified 2035 mouse and 1847 human genes that utilize substantially distal novel 3′ UTRs. Each of these extends at least 500 bases past the most distal 3′ termini available in Ensembl v65, and collectively they add 6.6 Mb and 5.1 Mb to the mRNA space of mouse and human, respectively. Extensive Northern analyses validated stable accumulation of distal APA isoforms, including transcripts bearing exceptionally long 3′ UTRs (many >10 kb and some >18 kb in length). The Northern data further illustrate that the extensions we annotated were not due to unprocessed transcriptional run-off events. Global tissue comparisons revealed that APA events yielding these extensions were most prevalent in the mouse and human brain. Finally, these extensions collectively contain thousands of conserved miRNA binding sites, and these are strongly enriched for many well-studied neural miRNAs. Altogether, these new 3′ UTR annotations greatly expand the scope of post-transcriptional regulatory networks in mammals, and have particular impact on the central nervous system.

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.

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150Glued by live-cell imaging. Time-lapse analysis of p150Glued revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.

SUMMARY We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3′ untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3′ UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3′ UTR length in the nervous system.

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.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.DOI: http://dx.doi.org/10.7554/eLife.26464.001

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.