Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin’s stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin’s stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin’s constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin’s stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.

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Severe muscle wasting and denervation in mice lacking the RNA-binding protein ZFP106

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608423113 ◽

2016 ◽

Vol 113 (31) ◽

pp. E4494-E4503 ◽

Cited By ~ 16

Author(s):

Douglas M. Anderson ◽

Jessica Cannavino ◽

Hui Li ◽

Kelly M. Anderson ◽

Benjamin R. Nelson ◽

...

Keyword(s):

Skeletal Muscle ◽

Motor Neurons ◽

Knockout Mice ◽

Muscle Wasting ◽

Rna Binding ◽

Neuromuscular Junctions ◽

Binding Protein ◽

Rna Binding Protein ◽

Binding Motif ◽

Nerve Muscle

Innervation of skeletal muscle by motor neurons occurs through the neuromuscular junction, a cholinergic synapse essential for normal muscle growth and function. Defects in nerve–muscle signaling cause a variety of neuromuscular disorders with features of ataxia, paralysis, skeletal muscle wasting, and degeneration. Here we show that the nuclear zinc finger protein ZFP106 is highly enriched in skeletal muscle and is required for postnatal maintenance of myofiber innervation by motor neurons. Genetic disruption of Zfp106 in mice results in progressive ataxia and hindlimb paralysis associated with motor neuron degeneration, severe muscle wasting, and premature death by 6 mo of age. We show that ZFP106 is an RNA-binding protein that associates with the core splicing factor RNA binding motif protein 39 (RBM39) and localizes to nuclear speckles adjacent to spliceosomes. Upon inhibition of pre-mRNA synthesis, ZFP106 translocates with other splicing factors to the nucleolus. Muscle and spinal cord of Zfp106 knockout mice displayed a gene expression signature of neuromuscular degeneration. Strikingly, altered splicing of the Nogo (Rtn4) gene locus in skeletal muscle of Zfp106 knockout mice resulted in ectopic expression of NOGO-A, the neurite outgrowth factor that inhibits nerve regeneration and destabilizes neuromuscular junctions. These findings reveal a central role for Zfp106 in the maintenance of nerve–muscle signaling, and highlight the involvement of aberrant RNA processing in neuromuscular disease pathogenesis.

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Exon Selection in α-Tropomyosin mRNA Is Regulated by the Antagonistic Action of RBM4 and PTB

Molecular and Cellular Biology ◽

10.1128/mcb.25.22.10111-10121.2005 ◽

2005 ◽

Vol 25 (22) ◽

pp. 10111-10121 ◽

Cited By ~ 53

Author(s):

Jung-Chun Lin ◽

Woan-Yuh Tarn

Keyword(s):

Skeletal Muscle ◽

Rna Binding ◽

Binding Motif ◽

Splicing Regulators ◽

Polypyrimidine Tract Binding Protein ◽

Muscle Cell Type ◽

Differential Display Analysis ◽

Splicing Regulator ◽

Mrna Isoform

ABSTRACT RNA-binding motif protein 4 (RBM4) has been implicated in the regulation of precursor mRNA splicing. Using differential display analysis, we identified mRNAs that associate with RBM4-containing messenger RNPs in vivo. Among these mRNAs, α-tropomyosin (α-TM) is known to exhibit a muscle cell type-specific splicing pattern. The level of the skeletal muscle-specific α-TM mRNA isoform partially correlated with that of RBM4 in human tissues examined and could be modulated by ectopic overexpression or suppression of RBM4. These results indicated that RBM4 directly influences the expression of the skeletal muscle-specific α-TM isoform. Using minigenes, we demonstrated that RBM4 can activate the selection of skeletal muscle-specific exons, possibly via binding to intronic pyrimidine-rich elements. By contrast, the splicing regulator polypyrimidine tract binding protein (PTB) excluded these exons; moreover, RBM4 antagonized this PTB-mediated exon exclusion likely by competing with PTB for binding to a CU-rich element. This study suggests a possible mechanism underlying the regulated alternative splicing of α-TM by the antagonistic splicing regulators RBM4 and PTB.

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Megaesophagus Is a Major Pathological Condition in Rats With a Large Deletion in the Rbm20 Gene

Veterinary Pathology ◽

10.1177/0300985819854224 ◽

2019 ◽

Vol 57 (1) ◽

pp. 151-159

Author(s):

Denise J. Schwahn ◽

Jonathan M. Pleitner ◽

Marion L. Greaser

Keyword(s):

Skeletal Muscle ◽

Rna Binding ◽

Pathological Condition ◽

Specific Protein ◽

Large Deletion ◽

Nasal Discharge ◽

Binding Motif ◽

Loss Of Function ◽

Unexpected Death ◽

Rat Strain

A spontaneously arising, loss-of-function mutation in the RNA binding motif protein 20 ( Rbm20) gene, which encodes a nuclear splicing protein, was previously identified as the underlying reason for expression of an abnormally large TITIN (TTN) protein in a rat model of cardiomyopathy. An outbreak of Pseudomonas aeruginosa led to submission of rats with dyspnea, sneezing, lethargy, nasal discharge, and/or unexpected death for diagnostic evaluation. Necropsy revealed underlying megaesophagus in Rbm20–/– rats. Further phenotyping of this rat strain and determination of the size of esophageal TTN was undertaken. The Rbm20-defective rats developed megaesophagus at an early age (26 weeks) with high frequency (13/32, 41%). They also often exhibited secondary rhinitis (9/32, 28%), aspiration pneumonia (8/32, 25%), and otitis media/interna (6/32, 19%). In addition, these rats had a high prevalence of hydronephrosis (13/32, 41%). RBM20 is involved in splicing multiple RNA transcripts, one of which is the muscle-specific protein TTN. Rbm20 mutations are a significant cause of dilated cardiomyopathy in humans. In Rbm20-defective rats, TTN size was significantly increased in the skeletal muscle of the esophagus. Megaesophagus in this rat strain (maintained on a mixed genetic background) is hypothesized to result from altered TTN stretch signaling in esophageal skeletal muscle. This study describes a novel mechanism for the development of megaesophagus, which may be useful for understanding the pathogenesis of megaesophagus in humans and offers insights into potential myogenic causes of this condition. This is the first report of megaesophagus and other noncardiac pathogenic changes associated with mutation of Rbm20 in any species.

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The Combined Human Genotype of Truncating TTN and RBM20 Mutations Is Associated with Severe and Early Onset of Dilated Cardiomyopathy

Genes ◽

10.3390/genes12060883 ◽

2021 ◽

Vol 12 (6) ◽

pp. 883

Author(s):

Anna Gaertner ◽

Julia Bloebaum ◽

Andreas Brodehl ◽

Baerbel Klauke ◽

Katharina Sielemann ◽

...

Keyword(s):

Heart Failure ◽

Dilated Cardiomyopathy ◽

Early Onset ◽

Target Genes ◽

Frameshift Mutation ◽

Rna Binding ◽

Splicing Factor ◽

Binding Motif ◽

Rich Domain ◽

Generation Sequencing

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.

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RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽

10.1038/s41388-021-01666-z ◽

2021 ◽

Author(s):

Qiuxia Yan ◽

Peng Zeng ◽

Xiuqin Zhou ◽

Xiaoying Zhao ◽

Runqiang Chen ◽

...

Keyword(s):

Bladder Cancer ◽

Alternative Splicing ◽

Rna Binding ◽

Aerobic Glycolysis ◽

Histological Grade ◽

Migration And Invasion ◽

Binding Motif ◽

Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

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Lack of Skeletal Muscle Serotonin Impairs Physical Performance

International Journal of Tryptophan Research ◽

10.1177/11786469211003109 ◽

2021 ◽

Vol 14 ◽

pp. 117864692110031

Author(s):

Marion Falabrègue ◽

Anne-Claire Boschat ◽

Romain Jouffroy ◽

Marieke Derquennes ◽

Haidar Djemai ◽

...

Keyword(s):

Skeletal Muscle ◽

Endurance Training ◽

Physical Performance ◽

Depressive Disorders ◽

Tryptophan Metabolism ◽

Long Distance ◽

Positive Effects ◽

Tryptophan Metabolites ◽

The Brain ◽

Deficient Mice

Low levels of the neurotransmitter serotonin have been associated with the onset of depression. While traditional treatments include antidepressants, physical exercise has emerged as an alternative for patients with depressive disorders. Yet there remains the fundamental question of how exercise is sensed by the brain. The existence of a muscle–brain endocrine loop has been proposed: according to this scenario, exercise modulates metabolization of tryptophan into kynurenine within skeletal muscle, which in turn affects the brain, enhancing resistance to depression. But the breakdown of tryptophan into kynurenine during exercise may also alter serotonin synthesis and help limit depression. In this study, we investigated whether peripheral serotonin might play a role in muscle–brain communication permitting adaptation for endurance training. We first quantified tryptophan metabolites in the blood of 4 trained athletes before and after a long-distance trail race and correlated changes in tryptophan metabolism with physical performance. In parallel, to assess exercise capacity and endurance in trained control and peripheral serotonin–deficient mice, we used a treadmill incremental test. Peripheral serotonin–deficient mice exhibited a significant drop in physical performance despite endurance training. Brain levels of tryptophan metabolites were similar in wild-type and peripheral serotonin–deficient animals, and no products of muscle-induced tryptophan metabolism were found in the plasma or brains of peripheral serotonin–deficient mice. But mass spectrometric analyses revealed a significant decrease in levels of 5-hydroxyindoleacetic acid (5-HIAA), the main serotonin metabolite, in both the soleus and plantaris muscles, demonstrating that metabolization of tryptophan into serotonin in muscles is essential for adaptation to endurance training. In light of these findings, the breakdown of tryptophan into peripheral but not brain serotonin appears to be the rate-limiting step for muscle adaptation to endurance training. The data suggest that there is a peripheral mechanism responsible for the positive effects of exercise, and that muscles are secretory organs with autocrine-paracrine roles in which serotonin has a local effect.

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RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

Nature Communications ◽

10.1038/s41467-021-21828-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xia Wang ◽

Jin Wang ◽

Yu-Man Tsui ◽

Chaoran Shi ◽

Ying Wang ◽

...

Keyword(s):

Stem Cells ◽

Mrna Stability ◽

Rna Binding ◽

Embryonic Stem ◽

Molecular Characteristics ◽

Specific Gene ◽

Functional Studies ◽

Poor Differentiation ◽

Development In Vitro

AbstractGrowing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

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Podocalyxin-like and RNA-binding motif protein 3 are prognostic biomarkers in urothelial bladder cancer: a validatory study

Biomarker Research ◽

10.1186/s40364-017-0090-y ◽

2017 ◽

Vol 5 (1) ◽

Cited By ~ 9

Author(s):

Karolina Boman ◽

Gustav Andersson ◽

Christoffer Wennersten ◽

Björn Nodin ◽

Göran Ahlgren ◽

...

Keyword(s):

Bladder Cancer ◽

Rna Binding ◽

Prognostic Biomarkers ◽

Binding Motif ◽

Urothelial Bladder Cancer ◽

Urothelial Bladder

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Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

The Journal of General Physiology ◽

10.1085/jgp.200709790 ◽

2007 ◽

Vol 130 (4) ◽

pp. 365-378 ◽

Cited By ~ 57

Author(s):

Sanjeewa A. Goonasekera ◽

Nicole A. Beard ◽

Linda Groom ◽

Takashi Kimura ◽

Alla D. Lyfenko ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Calcium Release ◽

Striated Muscle ◽

Single Channel ◽

Triple Mutant ◽

Double Mutation ◽

Excitation Contraction Coupling ◽

Contraction Coupling ◽

Functional Studies

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

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