RESUMEN
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Asunto(s)
Reparación del ADN/fisiología , ARN Polimerasa II/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN/metabolismo , Daño del ADN/fisiología , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Polimerasa II/genética , UbiquitinaciónRESUMEN
The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.
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Núcleo Celular , Empalme del ARN , Proteínas de Unión al ARN , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Precursores del ARN/metabolismo , Precursores del ARN/genética , Mutación , Empalmosomas/metabolismo , Empalmosomas/genética , Células HeLa , Células HEK293RESUMEN
Although large exons cannot be readily recognized by the spliceosome, many are evolutionarily conserved and constitutively spliced for inclusion in the processed transcript. Furthermore, whether large exons may be enriched in a certain subset of proteins, or mediate specific functions, has remained unclear. Here, we identify a set of nearly 3,000 SRSF3-dependent large constitutive exons (S3-LCEs) in human and mouse cells. These exons are enriched for cytidine-rich sequence motifs, which bind and recruit the splicing factors hnRNP K and SRSF3. We find that hnRNP K suppresses S3-LCE splicing, an effect that is mitigated by SRSF3 to thus achieve constitutive splicing of S3-LCEs. S3-LCEs are enriched in genes for components of transcription machineries, including mediator and BAF complexes, and frequently contain intrinsically disordered regions (IDRs). In a subset of analyzed S3-LCE-containing transcription factors, SRSF3 depletion leads to deletion of the IDRs due to S3-LCE exon skipping, thereby disrupting phase-separated assemblies of these factors. Cytidine enrichment in large exons introduces proline/serine codon bias in intrinsically disordered regions and appears to have been evolutionarily acquired in vertebrates. We propose that layered splicing regulation by hnRNP K and SRSF3 ensures proper phase-separation of these S3-LCE-containing transcription factors in vertebrates.
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Exones , Factores de Empalme Serina-Arginina/genética , Factores de Transcripción/genética , Vertebrados/genética , Animales , Línea Celular , Citidina/genética , Evolución Molecular , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Poliadenilación , Empalme del ARN , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative ß-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where ß-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where ß-catenin serves as a transcriptional coactivator in Wnt/ß-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/ß-catenin signaling that regulate the formation of the embryonic NMJ.
Asunto(s)
Transcriptoma , beta Catenina , Ratones , Animales , beta Catenina/metabolismo , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Vía de Señalización Wnt/genética , ARN Nuclear Pequeño/metabolismo , Desarrollo Embrionario , Músculo Esquelético/metabolismo , Receptores Colinérgicos/metabolismoRESUMEN
Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 µM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission.
RESUMEN
More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities.
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Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/metabolismo , ARN/metabolismo , Animales , Línea Celular Tumoral , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Poliadenilación , Unión Proteica , ARN Polimerasa II/metabolismo , TranscriptomaRESUMEN
Agrin is a heparan sulfate proteoglycan essential for the clustering of acetylcholine receptors at the neuromuscular junction. Neuron-specific isoforms of agrin are generated by alternative inclusion of three exons, called Y, Z8, and Z11 exons, although their processing mechanisms remain elusive. We found, by inspection of splicing cis-elements into the human AGRN gene, that binding sites for polypyrimidine tract binding protein 1 (PTBP1) were extensively enriched around Y and Z exons. PTBP1-silencing enhanced the coordinated inclusion of Y and Z exons in human SH-SY5Y neuronal cells, even though three constitutive exons are flanked by these alternative exons. Deletion analysis using minigenes identified five PTBP1-binding sites with remarkable splicing repression activities around Y and Z exons. Furthermore, artificial tethering experiments indicated that binding of a single PTBP1 molecule to any of these sites represses nearby Y or Z exons as well as the other distal exons. The RRM4 domain of PTBP1, which is required for looping out a target RNA segment, was likely to play a crucial role in the repression. Neuronal differentiation downregulates PTBP1 expression and promotes the coordinated inclusion of Y and Z exons. We propose that the reduction in the PTPB1-RNA network spanning these alternative exons is essential for the generation of the neuron-specific agrin isoforms.
Asunto(s)
Neuroblastoma , ARN , Humanos , ARN/metabolismo , Agrina/genética , Agrina/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismoRESUMEN
OBJECTIVES: Stroke patients frequently exhibit loss of independence of urination, and their lower urinary tract symptoms change with the phase of stroke. However, it is unclear whether switching prescribed drugs for lower urinary tract symptoms during hospitalization from acute care wards to convalescence rehabilitation wards affects patients' independence of urination at discharge. It is also unclear whether the impact of switching varies by stroke type. This retrospective cohort study aimed to examine these issues. MATERIALS AND METHODS: We analyzed 990 patients registered in the Kaga Regional Cooperation Clinical Pathway for Stroke database during 2015-2019. Prescriptions for lower urinary tract symptoms from pre-onset to convalescence rehabilitation were surveyed. Logistic regression analysis was performed to examine the association between switching drugs and independence of urination based on bladder management and voiding location at discharge. Stroke types were also examined in subgroup analyses. RESULTS: About 21 % of patients had their lower urinary tract symptoms prescriptions switched during hospitalization. Switching was positively associated with independence of bladder management (odds ratio 1.65, 95 % confidence interval 1.07 to 2.49) and voiding location (odds ratio 2.72, 95 % confidence interval 1.72 to 4.37). Similar associations were observed in different stroke types. CONCLUSIONS: Approximately 20 % of patients had their lower urinary tract symptoms medications switched upon transfer from acute to convalescence rehabilitation wards. Switching was significantly associated with improved urinary independence at discharge. Consistent results were observed across different stroke types, suggesting that switching medications contributes to urinary independence after stroke, regardless of the etiology or severity of stroke.
Asunto(s)
Síntomas del Sistema Urinario Inferior , Rehabilitación de Accidente Cerebrovascular , Accidente Cerebrovascular , Humanos , Micción , Convalecencia , Estudios Retrospectivos , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/complicaciones , Síntomas del Sistema Urinario Inferior/diagnóstico , Síntomas del Sistema Urinario Inferior/tratamiento farmacológico , Síntomas del Sistema Urinario Inferior/etiologíaRESUMEN
We screened pre-approved drugs for the survival of the Hu5/KD3 human myogenic progenitors. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, promoted the proliferation and survival of Hu5/KD3 cells. Meclozine increased expression of MyoD, but reduced expression of myosin heavy chain and suppressed myotube formation. Withdrawal of meclozine, however, resumed the ability of Hu5/KD3 cells to differentiate into myotubes. We examined the effects of meclozine on mdx mouse carrying a nonsense mutation in the dystrophin gene and modeling for Duchenne muscular dystrophy. Intragastric administration of meclozine in mdx mouse increased the body weight, the muscle mass in the lower limbs, the cross-sectional area of the paravertebral muscle, and improved exercise performances. Previous reports show that inhibition of phosphorylation of ERK1/2 improves muscle functions in mouse models for Emery-Dreifuss muscular dystrophy and cancer cachexia, as well as in mdx mice. We and others previously showed that meclozine blocks the phosphorylation of ERK1/2 in cultured cells. We currently showed that meclozine decreased phosphorylation of ERK1/2 in muscles in mdx mice but not in wild-type mice. This was likely to be one of the underlying mechanisms of the effects of meclozine on mdx mice.
Asunto(s)
Meclizina/farmacología , Fuerza Muscular/fisiología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Masculino , Meclizina/uso terapéutico , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Actividad Motora/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Fosforilación/efectos de los fármacosRESUMEN
RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.
Asunto(s)
Poliadenilación , Proteína FUS de Unión a ARN , Ribonucleoproteína Nuclear Pequeña U1 , Humanos , ARN/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismoRESUMEN
At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.
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Factor de Crecimiento del Tejido Conjuntivo , Proteínas Relacionadas con Receptor de LDL , Agrina/genética , Agrina/metabolismo , Animales , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Ratones , Unión Neuromuscular/metabolismo , FosforilaciónRESUMEN
During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other's activity, indicating an essential role of protein-RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein-RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein-RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein-RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein-RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.
Asunto(s)
Inmunoprecipitación/métodos , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Perfilación de la Expresión Génica , Humanos , Inmunoprecipitación/tendencias , Unión Proteica , ARN Polimerasa II/metabolismo , Transcripción Genética , TranscriptomaRESUMEN
OBJECTIVE: Reversible myelin vacuolization is associated with variable conditions including mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), which is characterized by mildly impaired consciousness and transient splenial lesion. Familial and/or recurrent cases with a clinical diagnosis of MERS suggest the presence of genetic factors. METHODS: We examined a family in which the proband presented with a history of recurrent encephalopathy with extensive but reversible cerebral myelin vacuolization and neurological symptoms similar to those of MERS spanning 3 generations. Whole-exome sequencing was performed in family members. RESULTS: Eight rare nonsynonymous single-nucleotide variants shared by all patients were identified. By filtering genes expressed in the corpus callosum, we identified a heterozygous c.1208A>G predicting p.Gln403Arg in the highly conserved DNA-binding domain in the myelin regulatory factor (MYRF) gene. We subsequently screened the coding regions of MYRF by Sanger sequencing in our cohort comprised of 33 sporadic cases with MERS and 3 cases in another family with extensive myelin vacuolization, and identified the same heterozygous c.1208A>G in all affected members in the second family. Luciferase assay revealed that transcriptional activity of the N-terminal region of MYRF was significantly diminished by introducing the c.1208A>G variant. INTERPRETATION: MYRF is a transcriptional regulator that is necessary for oligodendrocyte differentiation and myelin maintenance. Functional defects of MYRF are likely to be causally associated with encephalopathy with extensive myelin vacuolization. We propose the term "MYRF-related mild encephalopathy with reversible myelin vacuolization." Our findings provide a new perspective on the pathogenesis of myelin vacuolization. Ann Neurol 2018;83:98-106.
Asunto(s)
Encefalopatías/genética , Proteínas de la Membrana/genética , Vaina de Mielina/patología , Factores de Transcripción/genética , Adolescente , Adulto , Encefalopatías/diagnóstico por imagen , Encefalopatías/patología , Niño , Preescolar , Estudios de Cohortes , Cuerpo Calloso/metabolismo , Progresión de la Enfermedad , Electroencefalografía , Exoma/genética , Familia , Humanos , Imagen por Resonancia Magnética , Masculino , Linaje , Polimorfismo de Nucleótido Simple/genética , Vacuolas/patología , Adulto JovenRESUMEN
Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3Î end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3Î splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3Î ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3Î ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3Î ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.
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Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Empalme Alternativo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Poliadenilación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Unión Competitiva , Células CACO-2 , Línea Celular , Factor de Estimulación del Desdoblamiento , Exones , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/antagonistas & inhibidores , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos Reguladores de la TranscripciónRESUMEN
Right-sided infective endocarditis (IE) accounts for 3-14% of all cases of IE. Compared with leftsided IE, its antibiotic treatment is more effective. Therefore, the timing of its surgical treatment is still controversial. We report 2 cases of tricuspid valve IE and ventricular septal defect (VSD) associated with multiple lung abscesses and infarctions. After successful antibiotic treatment, they underwent vegetectomy, tricuspid valve plasty and VSD patch closure. Antibacterial treatment preceding surgical treatment is effective for tricuspid endocarditis complicated with multiple lung abscesses.
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Antibacterianos/uso terapéutico , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/cirugía , Defectos del Tabique Interventricular/cirugía , Enfermedades de las Válvulas Cardíacas/tratamiento farmacológico , Enfermedades de las Válvulas Cardíacas/cirugía , Absceso Pulmonar/tratamiento farmacológico , Válvula Tricúspide , Endocarditis Bacteriana/complicaciones , Defectos del Tabique Interventricular/complicaciones , Enfermedades de las Válvulas Cardíacas/complicaciones , Humanos , Absceso Pulmonar/complicacionesRESUMEN
We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET , AChEH , and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
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Colinérgicos/farmacología , Exones/genética , Síndromes Miasténicos Congénitos/genética , Unión Neuromuscular/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Empalme del ARN/efectos de los fármacos , Animales , Colinérgicos/metabolismo , Humanos , Unión Neuromuscular/genética , Empalme del ARN/genéticaRESUMEN
Proper bioriented attachment of microtubules and kinetochores is essential for the precise distribution of duplicated chromosomes to each daughter cell. An aberrant kinetochore-microtubule attachment results in chromosome instability, which leads to cellular transformation or apoptosis. In this article, we show that ubiquitin-associated protein 2-like (UBAP2L) is necessary for correct kinetochore-microtubule attachment. Depletion of UBAP2L inhibited chromosome alignment in metaphase and delayed progression to anaphase by activating spindle assembly checkpoint signaling. In addition, UBAP2L knockdown increased side-on attachment of kinetochores along the microtubules and suppressed stable kinetochore fiber formation. A proteomics analysis identified protein arginine methyltransferase (PRMT)1 as a direct interaction partner of UBAP2L. UBAP2L has an arginine- and glycine-rich motif called the RGG/RG or GAR motif in the N terminus. Biochemical analysis confirmed that arginine residues in the RGG/RG motif of UBAP2L were directly methylated by PRMT1. Finally, we demonstrated that the RGG/RG motif of UBAP2L is essential for the proper alignment of chromosomes in metaphase for the accurate distribution of chromosomes. Our results show a possible role for arginine methylation in UBAP2L for the progression of mitosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Cinetocoros/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Arginina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células HEK293 , Células HeLa , Humanos , Metilación , Microtúbulos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5' splice site can be blocked by SSOs to activate the exon. CONCLUSIONS: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oligonucleótidos/metabolismo , Empalme del ARN/genética , Células A549 , Secuencia de Bases , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Exones/genética , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Inmunoprecipitación , Modelos Biológicos , Nucleótidos/genética , Sitios de Empalme de ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcriptoma/genéticaRESUMEN
A 32-year-old woman was found to have a gastric adenocarcinoma with multiple bone metastases. Chemotherapy in the first, second and third-line was not effective. Blood examinations showed disseminated intravascular coagulation(DIC)at the end of the second-line chemotherapy. The fourth-line chemotherapy, infusional 5-fluorouracil and levofolinate calcium was performed. This resulted in a good response for DIC. This palliative therapy was effective and safety.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Coagulación Intravascular Diseminada/etiología , Neoplasias Gástricas/tratamiento farmacológico , Adenocarcinoma/complicaciones , Adenocarcinoma/cirugía , Adulto , Femenino , Fluorouracilo/administración & dosificación , Gastrectomía , Humanos , Levoleucovorina/administración & dosificación , Neoplasias Gástricas/complicaciones , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Resultado del TratamientoRESUMEN
Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani-Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner.