Alternative Splicing Regulates the Physiological Adaptation of the Mouse Hind Limb Postural and Phasic Muscles to Microgravity

Muscle atrophy and fiber type alterations are well-characterized physiological adaptations to microgravity with both understood to be primarily regulated by differential gene expression (DGE). While microgravity-induced DGE has been extensively investigated, adaptations to microgravity due to alternative splicing (AS) have not been studied in a mammalian model. We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both DGE and changes in AS due to extended spaceflight. Tissue sections and total RNA were isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. Immunohistochemistry disclosed muscle type-specific physiological adaptations to microgravity that included i) a pronounced reduction in muscle fiber cross-sectional area in both muscles and ii) a prominent slow-to-fast fiber type transition in the gastrocnemius. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with lipid metabolism and mitochondrial function. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

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Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition

Nature Communications ◽

10.1038/s41467-021-20979-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

E. Agirre ◽

A. J. Oldfield ◽

N. Bellora ◽

A. Segelle ◽

R. F. Luco

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Embryonic Stem ◽

Chromatin Modifications ◽

Histone Marks ◽

Splicing Regulators ◽

Machine Learning Approach ◽

Alternatively Spliced ◽

Rna Motif ◽

Regulation Changes

AbstractAlternative splicing relies on the combinatorial recruitment of splicing regulators to specific RNA binding sites. Chromatin has been shown to impact this recruitment. However, a limited number of histone marks have been studied at a global level. In this work, a machine learning approach, applied to extensive epigenomics datasets in human H1 embryonic stem cells and IMR90 foetal fibroblasts, has identified eleven chromatin modifications that differentially mark alternatively spliced exons depending on the level of exon inclusion. These marks act in a combinatorial and position-dependent way, creating characteristic splicing-associated chromatin signatures (SACS). In support of a functional role for SACS in coordinating splicing regulation, changes in the alternative splicing of SACS-marked exons between ten different cell lines correlate with changes in SACS enrichment levels and recruitment of the splicing regulators predicted by RNA motif search analysis. We propose the dynamic nature of chromatin modifications as a mechanism to rapidly fine-tune alternative splicing when necessary.

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Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

Life Science Alliance ◽

10.26508/lsa.202101342 ◽

2022 ◽

Vol 5 (4) ◽

pp. e202101342

Author(s):

Elena Nikonova ◽

Amartya Mukherjee ◽

Ketaki Kamble ◽

Christiane Barz ◽

Upendra Nongthomba ◽

...

Keyword(s):

Alternative Splicing ◽

Muscle Development ◽

Rna Binding ◽

Troponin I ◽

Rna Binding Proteins ◽

Fiber Type ◽

Structural Defects ◽

Specific Gene ◽

Fiber Types ◽

Isoform Expression

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.

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hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

10.21203/rs.3.rs-1060705/v1 ◽

2021 ◽

Author(s):

Shuiqiao Yuan ◽

Shenglei Feng ◽

Jinmei Li ◽

Hui Wen ◽

Kuan Liu ◽

...

Keyword(s):

Alternative Splicing ◽

Germ Cell ◽

Germ Cells ◽

Rna Binding ◽

Cell Development ◽

Mrna Splicing ◽

Conditional Knockout ◽

Cell Junction ◽

Germ Cell Development ◽

Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

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Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Journal of Biological Chemistry ◽

10.1074/jbc.m116.748335 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3466-3480 ◽

Cited By ~ 24

Author(s):

Jonàs Juan-Mateu ◽

Tatiana H. Rech ◽

Olatz Villate ◽

Esther Lizarraga-Mollinedo ◽

Anna Wendt ◽

...

Keyword(s):

Alternative Splicing ◽

Beta Cell ◽

Beta Cells ◽

Insulin Release ◽

Beta Cell Function ◽

Rna Binding ◽

Cell Function ◽

Rna Binding Proteins ◽

Pancreatic Beta Cell ◽

Splicing Regulators

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-βH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.

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Physiological Adaptations following Resistance Training in Youth Athletes—A Narrative Review

Pediatric Exercise Science ◽

10.1123/pes.2016-0023 ◽

2016 ◽

Vol 28 (4) ◽

pp. 501-520 ◽

Cited By ~ 26

Author(s):

Kirsten Legerlotz ◽

Robert Marzilger ◽

Sebastian Bohm ◽

Adamantios Arampatzis

Keyword(s):

Resistance Training ◽

Injury Risk ◽

Structural Changes ◽

Neuromuscular Control ◽

Physiological Adaptation ◽

Cross Sectional ◽

Youth Athletes ◽

Stimulus Response ◽

Physiological Adaptations ◽

Induced Changes

Purpose:To understand the mechanisms for the effects of resistance training on functional parameters, and to assess the injury risk of the involved tissues, it is necessary to examine the underlying morphological and structural changes of the respective tissues.Methods:The presented information on physiological adaptations have been deduced from cross-sectional studies comparing youth athletes with controls and children with adults as well as from longitudinal studies examining the effects of resistance training in untrained children and adolescents and in youth athletes.Results:The evidence indicates, that training induced changes in motor performance rely partly on enhanced neuromuscular control, and partly on morphological adaptation of muscles and tendons, such as changes in muscle, muscle fiber and tendon cross-sectional area, muscle composition, and tendon material properties, with the bone also adapting by increasing bone mineral content and cortical area.Conclusion:Although the training induced adaptations of the investigated tissues follows similar principles in children as in adults, the magnitude of the adaptive response appears to be more subtle. As studies investigating physiological adaptation in youth athletes are sparse, more research in this area is warranted to elucidate the specific physiological stimulus-response relationship necessary for effective training programs and injury prevention.

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A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

10.20944/preprints202108.0137.v1 ◽

2021 ◽

Author(s):

Shao-Yen Kao ◽

Elena Nikonova ◽

Sabrina Chaabane ◽

Albiona Sabani ◽

Alexandra Martitz ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Processing ◽

Muscle Development ◽

Rna Binding ◽

Flight Muscle ◽

Rna Binding Proteins ◽

Fiber Type ◽

Expression Patterns ◽

Fine Tuning ◽

Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability between muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a useful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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NOVA2-mediated RNA regulation is required for axonal pathfinding during development

eLife ◽

10.7554/elife.14371 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 52

Author(s):

Yuhki Saito ◽

Soledad Miranda-Rottmann ◽

Matteo Ruggiu ◽

Christopher Y Park ◽

John J Fak ◽

...

Keyword(s):

Alternative Splicing ◽

Axon Guidance ◽

Rna Binding ◽

Rna Binding Proteins ◽

Axonal Pathfinding ◽

Rna Regulation ◽

Splicing Regulators ◽

Mouse Cortex ◽

Alternative Splicing Events

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

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Rat locomotory muscle fiber activity during trotting and galloping

Journal of Applied Physiology ◽

10.1152/jappl.1978.44.3.358 ◽

1978 ◽

Vol 44 (3) ◽

pp. 358-363 ◽

Cited By ~ 50

Author(s):

T. E. Sullivan ◽

R. B. Armstrong

Keyword(s):

Rattus Norvegicus ◽

Hind Limb ◽

Fiber Type ◽

Cross Sectional Area ◽

Sectional Area ◽

Glycogen Depletion ◽

Active Muscle ◽

Cross Sectional ◽

Limb Muscles ◽

Whole Muscle

This study was designed to investigate the hypothesis that the trot-gallop transition in running guadrupeds occurs when active cross-sectional areas of muscles or fiber populations within muscles, reach some critical point as animals increase speed within trotting. Rats (Rattus norvegicus) were used as experimental animals, and glycogen depletion was used to estimate patterns or fiber activity. Our results indicate that 1) the contribution to power output by the front limb muscles was less than that of the hind limb muscles during trotting and galloping; 2) the active cross-sectional area of plantaris muscles peaked immediately prior to the transition in gait; 3) the ankle plantar flexor group of muscles as a whole did not attain a maximum active cross-sectional area during fast trotting; and 4) no major discontinuities in whole muscle or fiber type glycogen depletion rates occurred across the gait change. Although these findings do not prove the hypothesis, they support the concept that the trop-gallop transition follows the attainment of peak active muscle cross-sectional areas as animals increase trotting speed.

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Creatine depletion elicits structural, biochemical, and physiological adaptations in rat costal diaphragm

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.5.c1480 ◽

1996 ◽

Vol 271 (5) ◽

pp. C1480-C1486 ◽

Cited By ~ 9

Author(s):

S. Levine ◽

B. Tikunov ◽

D. Henson ◽

J. LaManca ◽

H. L. Sweeney

Keyword(s):

Fiber Type ◽

Competitive Inhibitor ◽

Control Group ◽

Cross Sectional ◽

Heavy Chains ◽

Physiological Adaptations ◽

Normal Rat ◽

Fiber Type Distribution ◽

Cr Depletion ◽

Initial Force

To elucidate adaptations elicited by creatine (Cr) depletion in the costal diaphragm (Dia), 16 12-wk-old male Fisher 344 rats had 2% beta-guanidinopropionic acid (beta-GPA), a competitive inhibitor of Cr transport into muscle, added to their food; a control group (Con) of 16 rats ate normal rat chow. After 18 wk, beta-GPA and Con Dia did not differ histochemically with respect to fiber-type distribution; however, the cross-sectional area of type II(b + x) fibers was 33% less in beta-GPA than Con Dia. Biochemically, the proportion of myosin heavy chain IIb in beta-GPA Dia was decreased 42% from Con Dia, whereas the proportions of myosin heavy chains I and IIa were increased. Physiologically, both peak twitch tension and tetanic tension in beta-GPA Dia were decreased 40% from Con. To assess fatigability, we used the protocol of Kelsen and Nochomovitz (J. Appl. Physiol. 53; 440-447, 1982) for 2-6 min duration; the percentage of initial force exhibited by beta-GPA Dia was approximately twice that of Con Dia. We conclude that these structural, biochemical, and physiological adaptations elicited by Cr depletion can all be explained by selective atrophy of IIb muscle fibers in the Dia.

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A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

Cells ◽

10.3390/cells10102505 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2505

Author(s):

Shao-Yen Kao ◽

Elena Nikonova ◽

Sabrina Chaabane ◽

Albiona Sabani ◽

Alexandra Martitz ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Processing ◽

Muscle Development ◽

Rna Binding ◽

Flight Muscle ◽

Rna Binding Proteins ◽

Fiber Type ◽

Expression Patterns ◽

Fine Tuning ◽

Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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