Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy.

The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.

Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: Application to the ascidian Ciona intestinalis.

Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We report a computational strategy that overcomes these difficulties, and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. We developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes.

Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene.

In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5'-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5'-leader trans-splicing (SL trans-splicing) because the original 5'-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5'-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA's original 5'-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla.

Pathogenic peptide deviations support a model of adaptive evolution of chordate cardiac performance by troponin mutations.

In cardiac muscle, the troponin (cTn) complex is a key regulator of myofilament calcium sensitivity because it serves as a molecular switch required for translating myocyte calcium fluxes into sarcomeric contraction and relaxation. Studies of several species suggest that ectotherm chordates have myofilaments with heightened calcium responsiveness. However, genetic polymorphisms in cTn that cause increased myofilament sensitivity to activating calcium in mammals result in cardiac disease including arrhythmias, diastolic dysfunction, and increased susceptibility to sudden cardiac death. We hypothesized that specific residue modifications in the regulatory arm of troponin I (TnI) were critical in mediating the observed decrease in myofilament calcium sensitivity within the mammalian taxa. We performed large-scale phylogenetic analysis, atomic resolution molecular dynamics simulations and modeling, and computational alanine scanning. This study provides evidence that a His to Ala substitution within mammalian cardiac TnI (cTnI) reduced the thermodynamic potential at the interface between cTnI and cardiac TnC (cTnC) in the calcium-saturated state by disrupting a strong intermolecular electrostatic interaction. This key residue modification reduced myofilament calcium sensitivity by making cTnI molecularly untethered from cTnC. To meet the requirements for refined mammalian adult cardiac performance, we propose that compensatory evolutionary pressures favored mutations that enhanced the relaxation properties of cTn by decreasing its sensitivity to activating calcium.

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

SL RNA genes of the ascidian tunicates Ciona intestinalis and Ciona savignyi.

We characterized by bioinformatics the trans-spliced leader donor RNA (SL RNA) genes of two ascidians, Ciona intestinalis and Ciona savignyi. The Ciona intestinalis genome contains approximately 670 copies of the SL RNA gene, principally on a 264-bp tandemly repeated element. Fluorescent in-situ hybridization mapped most of the repeats to a single site on the short arm of chromosome 8. The Ciona intestinalis genome also contains approximately 100 copies of a >3.6-kb element that carries 1) an SL RNA-related sequence (possible a pseudogene) and 2) genes for the U6 snRNA and a histone-like protein. The Ciona savignyi genome contains two SL RNA gene classes having the same SL sequence as Ciona intestinalis but differing in the intron-like segments. These reside in similar but distinct repeat units of 575 bp ( approximately 410 copies) and 552 bp ( approximately 250 copies) that are arranged as separate tandem repeats. In neither Ciona species is the 5S RNA gene present within the SL RNA gene repeat unit. Although the number of SL RNA genes is similar, there is little sequence similarity between the intestinalis and savignyi repeat units, apart from the region encoding the SL RNA itself. This suggests that cis-regulatory elements involved in transcription and 3'-end processing are likely to be present within the transcribed region. The genomes of both Ciona species also include > 100 dispersed short elements containing the 16-nt SL sequence and up to 6 additional nucleotides of the SL RNA sequence.

Strong muscle-specific regulatory cassettes based on multiple copies of the human slow troponin I gene upstream enhancer.

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice. These constructs were evaluated by measuring the activity of the reporter gene beta-galactosidase (beta-gal). In cell culture, these multimerized enhancers retained tissue specificity and their transcriptional strength increased with the number of enhancer copies. In myotube cultures (which still contain nondifferentiated cells), constructs containing four and five USE copies were stronger than the cytomegalovirus (CMV) early enhancer/promoter and slightly weaker than the hybrid CMV enhancer/beta-actin (CB) promoter. Those containing three USE, or four DeltaUSE copies were similar in strength to CMV. After electrotransfer of plasmid DNA into the mouse tibialis anterior muscle, the strengths of the two constructs (USEx3 and DeltaUSEx3) were tested; as measured by beta-gal activity in the total muscle lysate and by the number of transduced fibers, they were similar to CMV and CB. Muscle fiber typing, after electrotransfer of the soleus muscle, showed that DeltaUSEx3 and USEx3 were active in slow and fast fibers. The tissue specificity of these two constructs was also evaluated by hydrodynamic plasmid injection through the tail vein. Although significant beta-gal expression was measured in the liver when CMV was tested, no expression above background level was detected with USEx3 and DeltaUSEx3. The strength, muscle specificity, and small size of these transcriptional elements render them very attractive for gene therapy applications.

Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis: new insight into intron and operon populations.

BACKGROUND: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly. RESULTS: We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5'-ends were precisely mapped using 5'-full-length ESTs, an important refinement even in otherwise unchanged models. CONCLUSION: Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever.

Genomic overview of mRNA 5'-leader trans-splicing in the ascidian Ciona intestinalis.

Although spliced leader (SL) trans-splicing in the chordates was discovered in the tunicate Ciona intestinalis there has been no genomic overview analysis of the extent of trans-splicing or the make-up of the trans-spliced and non-trans-spliced gene populations of this model organism. Here we report such an analysis for Ciona based on the oligo-capping full-length cDNA approach. We randomly sampled 2078 5'-full-length ESTs representing 668 genes, or 4.2% of the entire genome. Our results indicate that Ciona contains a single major SL, which is efficiently trans-spliced to mRNAs transcribed from a specific set of genes representing approximately 50% of the total number of expressed genes, and that individual trans-spliced mRNA species are, on average, 2-3-fold less abundant than non-trans-spliced mRNA species. Our results also identify a relationship between trans-splicing status and gene functional classification; ribosomal protein genes fall predominantly into the non-trans-spliced category. In addition, our data provide the first evidence for the occurrence of polycistronic transcription in Ciona. An interesting feature of the Ciona polycistronic transcription units is that the great majority entirely lack intercistronic sequences.

SL trans-splicing: easy come or easy go?

Is spliced-leader (SL) trans-splicing an ancestral eukaryotic characteristic that has been lost in multiple lineages, or did it arise independently in the various phyla in which it occurs? Recent studies have discovered SL trans-splicing in new metazoan phyla, including the chordates. Its discovery in chordates identifies, for the first time, a phylum that clearly contains both trans-splicing and non-trans-splicing major groups, and defines a limited and well-understood field in which to study the evolutionary dynamics of SL trans-splicing. In this article, I summarize the evolutionarily relevant aspects of SL trans-splicing and consider the interplay among SL trans-splicing, pre-mRNA splice-signal syntax and evolutionary genomics.

Ascidian larva reveals ancient origin of vertebrate-skeletal-muscle troponin I characteristics in chordate locomotory muscle.

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle -like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. IX. Genes for muscle structural proteins.

Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types - larval tail muscle, adult body-wall muscle and heart - expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

TnIfast IRE enhancer: multistep developmental regulation during skeletal muscle fiber type differentiation.

To identify developmental steps leading to adult skeletal muscle fiber-type-specific gene expression, we carried out transgenic mouse studies of the IRE enhancer of the quail TnIfast gene. Histochemical analysis of IRE/herpesvirus tk promoter/beta-galactosidase reporter transgene expression in adult muscle directly demonstrated IRE-driven fast vs. slow fiber-type specificity, and IIB>IIX>IIA differential expression among the fast fiber types: patterns similar to those of native-promoter TnIfast constructs. These tissue- and cell-type specificities are autonomous to the IRE and do not depend on interactions with a muscle gene promoter. Developmental studies showed that the adult pattern of IRE-driven transgene expression emerges in three steps: (1) activation during the formation of primary embryonic (presumptive slow) muscle fibers; (2) activation, to markedly higher levels, during formation of secondary (presumptive fast) fibers, and (3) differential augmentation of expression during early postnatal maturation of the IIB, IIX, IIA fast fiber types. These results provide insight into the roles of gene activation and gene repression mechanisms in fiber-type specificity and can account for apparently disparate results obtained in previous studies of TnI isoform expression in development. Each of the three IRE-driven developmental steps is spatiotemporally associated with a different major regulatory event at the fast myosin heavy chain gene cluster, suggesting that diverse muscle gene families respond to common, or tightly integrated, regulatory signals during multiple steps of muscle fiber differentiation.

Coregulation of fast contractile protein transgene and glycolytic enzyme expression in mouse skeletal muscle.

Little is known of the gene regulatory mechanisms that coordinate the contractile and metabolic specializations of skeletal muscle fibers. Here we report a novel connection between fast isoform contractile protein transgene and glycolytic enzyme expression. In quantitative histochemical studies of transgenic mouse muscle fibers, we found extensive coregulation of the glycolytic enzyme glycerol-3-phosphate dehydrogenase (GPDH) and transgene constructs based on the fast skeletal muscle troponin I (TnIfast) gene. In addition to a common IIB > IIX > IIA fiber type pattern, TnIfast transgenes and GPDH showed correlated fiber-to-fiber variation within each fast fiber type, concerted emergence of high-level expression during early postnatal muscle maturation, and parallel responses to muscle under- or overloading. Regulatory information for GPDH-coregulated expression is carried by the TnIfast first-intron enhancer (IRE). These results identify an unexpected contractile/metabolic gene regulatory link that is amenable to further molecular characterization. They also raise the possibility that the equal expression in all fast fiber types observed for the endogenous TnIfast gene may be driven by different metabolically coordinated mechanisms in glycolytic (IIB) vs. oxidative (IIA) fast fibers.