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1.
Mol Cell ; 81(7): 1358-1362, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33798410

RESUMEN

We talk to the authors of "Structural basis for conformational equilibrium of the catalytic spliceosome" about their paper, advances in cryo-EM that made it possible, and the influence of their late mentor Kiyoshi Nagai, to whom the paper is dedicated.


Asunto(s)
Microscopía por Crioelectrón/historia , Empalme del ARN , Empalmosomas , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Retratos como Asunto
2.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-33802303

RESUMEN

Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cotiledón/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes del Cloroplasto/genética , Plastidios/genética , Cloroplastos , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética , Interferencia de ARN/fisiología , Empalme del ARN/genética , Ribosomas/genética , Plantones/genética
3.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805770

RESUMEN

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.


Asunto(s)
Conectina/genética , Músculo Esquelético/efectos de los fármacos , Mioblastos/efectos de los fármacos , Precursores del ARN/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Animales , Antitiroideos/farmacología , Línea Celular , Conectina/metabolismo , Cruzamientos Genéticos , Femenino , Humanos , Masculino , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Especificidad de Órganos , Propiltiouracilo/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Estreptozocina/farmacología , Triyodotironina/farmacología
4.
Nat Commun ; 12(1): 2300, 2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33863890

RESUMEN

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.


Asunto(s)
Emparejamiento Base/fisiología , ADN Complementario/genética , Precursores del ARN/metabolismo , Empalme del ARN/fisiología , Células A549 , Secuencia de Bases/genética , Secuencia Conservada/fisiología , Biblioteca de Genes , Células Hep G2 , Humanos , Intrones/genética , Poliadenilación , Pliegue del ARN/fisiología , Precursores del ARN/genética , RNA-Seq
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(3): 205-209, 2021 Mar 10.
Artículo en Chino | MEDLINE | ID: mdl-33751525

RESUMEN

OBJECTIVE: To analyze the clinical phenotype and genetic variants in five Chinese pedigrees affected with Dysferlinopathy. METHODS: Next generation sequencing (NGS) was carried out for the probands from the five pedigrees. Suspected variants were validated by Sanger sequencing. Pathogenicity of the variants was assessed based on the standards and guidelines by the American College of Medical Genetics and Genomics (ACMG). RESULTS: Ten DYSF gene variants (including 5 frameshift variants, 3 splicing variants, 1 missense variant and 1 nonsense variant) were detected. Among these, c.1375dupA (p.Met459Asnfs*15), c.610C>T (p.Arg204X), c.1180+5G>A and c.1284+2T>C were known to be pathogenic, while c.4008_4010delCCTinsAC (p.Leu1337Argfs*8), c.1137_1169del (p.379_390del), c.754A>G(p.Thr252Ala), c.1175_1176insGCAGAGTG (p.Met394Serfs*7), c.3114_3115insCGGC (p.Arg1040Profs*74) and c.1053+3G>C were unreported previously. Of the six novel variants, c.1137_1169del, c.1175_1176insGCAGAGTG and c.3114_3115insCGGC were predicted as pathogenic (PVS1+PM2+PM3), c.4008_4010delCCTinsAC as likely pathogenic (PVS1+PM2), c.754A>G and c.1053+3G>C as variants of uncertain significance based on the ACMG standards and guidelines. CONCLUSION: Variants of the DYSF gene probably underlay Dysferlinopathy in the patients among the five pedigrees. Above finding has enriched the spectrum of DYSF gene variants.


Asunto(s)
Distrofia Muscular de Cinturas , Empalme del ARN , Humanos , Distrofia Muscular de Cinturas/genética , Mutación , Linaje , Fenotipo
6.
Nat Commun ; 12(1): 1488, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674615

RESUMEN

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.


Asunto(s)
Exones , ARN Helicasas/química , ARN Helicasas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Modelos Moleculares , Precursores del ARN/química , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN de Hongos/metabolismo , Proteínas Recombinantes , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Empalmosomas/química
7.
Stem Cell Reports ; 16(3): 478-492, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33657418

RESUMEN

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.


Asunto(s)
/virología , Células Madre Pluripotentes Inducidas/virología , Miocitos Cardíacos/virología , /patogenicidad , Células Cultivadas , Humanos , Empalme del ARN/genética , ARN Mensajero/genética , Internalización del Virus
8.
Mol Cell ; 81(7): 1439-1452.e9, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33705709

RESUMEN

The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.


Asunto(s)
Modelos Químicos , Empalme del ARN , ARN de Hongos/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Empalmosomas/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo
9.
Nat Med ; 27(3): 526-535, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33707772

RESUMEN

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal childhood premature aging disorder caused by a pre-messenger RNA (mRNA) splicing defect in the LMNA gene. We used combined in vitro screening and in vivo validation to systematically explore the effects of target sequence, backbone chemistry and mechanism of action to identify optimized antisense oligonucleotides (ASOs) for therapeutic use in HGPS. In a library of 198 ASOs, the most potent ASOs targeted the LMNA exon 12 junction and acted via non-RNase H-mediated mechanisms. Treatment with an optimized lead candidate resulted in extension of lifespan in a mouse model of HGPS. Progerin mRNA levels were robustly reduced in vivo, but the extent of progerin protein reduction differed between tissues, suggesting a long half-life and tissue-specific turnover of progerin in vivo. These results identify a novel therapeutic agent for HGPS and provide insight into the HGPS disease mechanism.


Asunto(s)
Oligonucleótidos Antisentido/uso terapéutico , Progeria/tratamiento farmacológico , Humanos , Lamina Tipo A/genética , Prueba de Estudio Conceptual , Empalme del ARN
10.
Nat Commun ; 12(1): 1828, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758195

RESUMEN

DNA sequences containing consecutive guanines organized in 4-interspaced tandem repeats can form stable single-stranded secondary structures, called G-quadruplexes (G4). Herein, we report that the Polycomb group protein BMI1 is enriched at heterochromatin regions containing putative G4 DNA sequences, and that G4 structures accumulate in cells with reduced BMI1 expression and/or relaxed chromatin, including sporadic Alzheimer's disease (AD) neurons. In AD neurons, G4 structures preferentially accumulate in lamina-associated domains, and this is rescued by re-establishing chromatin compaction. ChIP-seq analyses reveal that G4 peaks correspond to evolutionary conserved Long Interspersed Element-1 (L1) sequences predicted to be transcriptionally active. Hence, G4 structures co-localize with RNAPII, and inhibition of transcription can reverse the G4 phenotype without affecting chromatin's state, thus uncoupling both components. Intragenic G4 structures affecting splicing events are furthermore associated with reduced neuronal gene expression in AD. Active L1 sequences are thus at the origin of most G4 structures observed in human neurons.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Eucromatina/metabolismo , G-Cuádruplex , Elementos de Nucleótido Esparcido Largo/genética , Neurogénesis/genética , Neuronas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Enfermedad de Alzheimer/genética , Animales , Células Cultivadas , Secuenciación de Inmunoprecipitación de Cromatina , Eucromatina/genética , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Ontología de Genes , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 1/genética , Empalme del ARN/genética
11.
Int J Mol Sci ; 22(5)2021 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-33668121

RESUMEN

The frameshift mutants K192Sfs*7 and R153Sfs*41, of the polyglutamine tract-binding protein 1 (PQBP-1), are stable intrinsically disordered proteins (IDPs). They are each associated with the severe cognitive disorder known as the Renpenning syndrome, a form of X-linked intellectual disability (XLID). Relative to the monomeric wild-type protein, these mutants are dimeric, contain more folded contents, and have higher thermal stabilities. Comparisons can be drawn to the toxic oligomerisation in the "conformational diseases", which collectively describe medical conditions involving a substantial protein structural transition in the pathogenic mechanism. At the molecular level, the end state of these diseases is often cytotoxic protein aggregation. The conformational disease proteins contain varying extents of intrinsic disorder, and the consensus pathogenesis includes an early oligomer formation. We reviewed the experimental characterisation of the toxic oligomers in representative cases. PQBP-1 mutant dimerisation was then compared to the oligomerisation of the conformational disease proteins. The PQBP-1 mutants are unique in behaving as stable soluble dimers, which do not further develop into higher oligomers or aggregates. The toxicity of the PQBP-1 mutant dimers lies in the native functions (in transcription regulation and possibly, RNA splicing) being compromised, rather than proceeding to aggregation. Other examples of stable IDP dimers were discussed and we speculated on the roles of IDP dimerisation in protein evolution.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Mutación del Sistema de Lectura , Genes Ligados a X , Discapacidad Intelectual/patología , Proteínas Mutantes/química , Proteínas de Unión al ADN/metabolismo , Humanos , Discapacidad Intelectual/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Multimerización de Proteína , Empalme del ARN
12.
Nat Commun ; 12(1): 1214, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33619278

RESUMEN

Melanoma is the most lethal skin malignancy, driven by genetic and epigenetic alterations in the complex tumour microenvironment. While large-scale molecular profiling of melanoma has identified molecular signatures associated with melanoma progression, comprehensive systems-level modeling remains elusive. This study builds up predictive gene network models of molecular alterations in primary melanoma by integrating large-scale bulk-based multi-omic and single-cell transcriptomic data. Incorporating clinical, epigenetic, and proteomic data into these networks reveals key subnetworks, cell types, and regulators underlying melanoma progression. Tumors with high immune infiltrates are found to be associated with good prognosis, presumably due to induced CD8+ T-cell cytotoxicity, via MYO1F-mediated M1-polarization of macrophages. Seventeen key drivers of the gene subnetworks associated with poor prognosis, including the transcription factor ZNF180, are tested for their pro-tumorigenic effects in vitro. The anti-tumor effect of silencing ZNF180 is further validated using in vivo xenografts. Experimentally validated targets of ZNF180 are enriched in the ZNF180 centered network and the known pathways such as melanoma cell maintenance and immune cell infiltration. The transcriptional networks and their critical regulators provide insights into the molecular mechanisms of melanomagenesis and pave the way for developing therapeutic strategies for melanoma.


Asunto(s)
Redes Reguladoras de Genes , Melanoma/patología , Modelos Biológicos , Neoplasias Cutáneas/patología , Microambiente Tumoral , Línea Celular Tumoral , Reparación del ADN , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Interferón gamma/metabolismo , Melanoma/genética , Miosina Tipo I/metabolismo , Invasividad Neoplásica , Pronóstico , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Neoplasias Cutáneas/genética , Análisis de Supervivencia , Microambiente Tumoral/genética , Regulación hacia Arriba/genética
13.
Nucleic Acids Res ; 49(5): 2509-2521, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33555349

RESUMEN

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.


Asunto(s)
Chaperonas Moleculares/genética , Proteínas Mutantes Quiméricas/genética , Neuroblastoma/genética , Empalme del ARN , Empalmosomas/efectos de los fármacos , Aminoaciltransferasas/metabolismo , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Femenino , Fusión Génica , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Ratones Desnudos , Chaperonas Moleculares/metabolismo , Proteínas Mutantes Quiméricas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Proteínas tau/metabolismo
14.
Nucleic Acids Res ; 49(5): 2552-2568, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33577675

RESUMEN

The meiotic gene expression program in Saccharomyces cerevisiae involves regulated splicing of meiosis-specific genes via multiple splicing activators (e.g. Mer1, Nam8, Tgs1). Here, we show that the SR protein Npl3 is required for meiotic splicing regulation and is essential for proper execution of the meiotic cell cycle. The loss of Npl3, though not required for viability in mitosis, caused intron retention in meiosis-specific transcripts, inefficient meiotic double strand break processing and an arrest of the meiotic cell cycle. The targets of Npl3 overlapped in some cases with other splicing regulators, while also having unique target transcripts that were not shared. In the absence of Npl3, splicing defects for three transcripts (MER2, HOP2 and SAE3) were rescued by conversion of non-consensus splice sites to the consensus sequence. Methylation of Npl3 was further found to be required for splicing Mer1-dependent transcripts, indicating transcript-specific mechanisms by which Npl3 supports splicing. Together these data identify an essential function for the budding yeast SR protein Npl3 in meiosis as part of the meiotic splicing regulatory network.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Meiosis/genética , Proteínas Nucleares/fisiología , Empalme del ARN , Proteínas de Unión al ARN/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Eliminación de Gen , Expresión Génica , Intrones , Metilación , Mitosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
N Engl J Med ; 384(10): 915-923, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33626251

RESUMEN

BACKGROUND: Type 1 spinal muscular atrophy is a rare, progressive neuromuscular disease that is caused by low levels of functional survival of motor neuron (SMN) protein. Risdiplam is an orally administered, small molecule that modifies SMN2 pre-messenger RNA splicing and increases levels of functional SMN protein. METHODS: We report the results of part 1 of a two-part, phase 2-3, open-label study of risdiplam in infants 1 to 7 months of age who had type 1 spinal muscular atrophy, which is characterized by the infant not attaining the ability to sit without support. Primary outcomes were safety, pharmacokinetics, pharmacodynamics (including the blood SMN protein concentration), and the selection of the risdiplam dose for part 2 of the study. Exploratory outcomes included the ability to sit without support for at least 5 seconds. RESULTS: A total of 21 infants were enrolled. Four infants were in a low-dose cohort and were treated with a final dose at month 12 of 0.08 mg of risdiplam per kilogram of body weight per day, and 17 were in a high-dose cohort and were treated with a final dose at month 12 of 0.2 mg per kilogram per day. The baseline median SMN protein concentrations in blood were 1.31 ng per milliliter in the low-dose cohort and 2.54 ng per milliliter in the high-dose cohort; at 12 months, the median values increased to 3.05 ng per milliliter and 5.66 ng per milliliter, respectively, which represented a median of 3.0 times and 1.9 times the baseline values in the low-dose and high-dose cohorts, respectively. Serious adverse events included pneumonia, respiratory tract infection, and acute respiratory failure. At the time of this publication, 4 infants had died of respiratory complications. Seven infants in the high-dose cohort and no infants in the low-dose cohort were able to sit without support for at least 5 seconds. The higher dose of risdiplam (0.2 mg per kilogram per day) was selected for part 2 of the study. CONCLUSIONS: In infants with type 1 spinal muscular atrophy, treatment with oral risdiplam led to an increased expression of functional SMN protein in the blood. (Funded by F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02913482.).


Asunto(s)
Compuestos Azo/administración & dosificación , Fármacos Neuromusculares/administración & dosificación , Pirimidinas/administración & dosificación , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Proteína 1 para la Supervivencia de la Neurona Motora/sangre , Administración Oral , Compuestos Azo/efectos adversos , Compuestos Azo/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lactante , Masculino , Fármacos Neuromusculares/efectos adversos , Fármacos Neuromusculares/farmacocinética , Supervivencia sin Progresión , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Empalme del ARN , Insuficiencia Respiratoria/etiología , Infecciones del Sistema Respiratorio/etiología , Atrofias Musculares Espinales de la Infancia/complicaciones , Atrofias Musculares Espinales de la Infancia/mortalidad , Proteína 1 para la Supervivencia de la Neurona Motora/genética
16.
Plant Mol Biol ; 105(6): 575-583, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33550520

RESUMEN

KEY MESSAGE: This review focused on the recent breakthroughs in plant high temperature responses from an alternative splicing angle. With the inevitable global warming, high temperature triggers plants to change their growth and developmental programs for adapting temperature increase. In the past decades, the signaling mechanisms from plant thermo-sensing to downstream transcriptional cascades have been extensively studied. Plenty of elegant review papers have summarized these breakthroughs from signal transduction to cross-talk within plant hormones and environmental cues. Precursor messenger RNA (pre-mRNA) splicing enables plants to produce a series of functional un-related proteins and thus enhances the regulation flexibility. Plants take advantage of this strategy to modulate their proteome diversity under high ambient temperature and elicit developmental plasticity. In this review, we particularly focus on pre-mRNA splicing regulation underlying plant high temperature responses, and will shed new light on the understanding of post-transcriptional regulation on plant growth and development.


Asunto(s)
Empalme Alternativo/fisiología , Calor , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme Alternativo/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica de las Plantas , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas , Empalme del ARN , ARN Mensajero/metabolismo , Temperatura
17.
Nat Commun ; 12(1): 1044, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594055

RESUMEN

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.


Asunto(s)
Bacteriófagos/genética , Microbioma Gastrointestinal/genética , Genoma Viral , Metagenoma , Codón/genética , Secuencia Conservada , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Inteínas , Intrones/genética , Sistemas de Lectura Abierta/genética , Filogenia , Empalme del ARN/genética , Transcripción Genética , /genética
18.
Viruses ; 13(2)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530363

RESUMEN

The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.


Asunto(s)
VIH-1/genética , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Empalme Alternativo , Exones , Regulación Viral de la Expresión Génica , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Secuencias Reguladoras de Ácidos Nucleicos , Latencia del Virus/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo
19.
Int J Mol Sci ; 22(4)2021 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-33546497

RESUMEN

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.


Asunto(s)
Amiloide/metabolismo , Codón sin Sentido , Priones/genética , Priones/metabolismo , Amiloidosis/genética , Amiloidosis/metabolismo , Amiloidosis/patología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Espectrometría de Masas , Priones/química , Agregado de Proteínas , Empalme del ARN
20.
Plant Physiol Biochem ; 160: 397-403, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33556755

RESUMEN

Environmental stresses activate endoplasmic reticulum (ER) stress response pathways, collectively known as the unfolded protein response (UPR). IRE1/bZIP60 pathway is the most conserved of all UPR pathways from yeast to plants. Transcription factor bZIP60 is activated by the cytoplasmic splicing of its mRNA by Inositol Requiring Enzyme1 (IRE1) protein. bZIP60 mRNA has a typical stem-loop structure that is required for its splicing by IRE1 ribonuclease. We identified the tomato bZIP60 (SlbZIP60) and secondary structure prediction showed that it has the conserved stem-loop structure. Further, we demonstrate that SlbZIP60 is spliced upon treatment with an ER stress-inducing agent, tunicamycin. Tunicamycin also upregulated the expression of SlbZIP60. Finally, we show that SlbZIP60 undergo physiologically activated splicing in certain tissues of the plant and respond to environmental stresses, heat, and virus infection. This study will help for a deeper understanding of ER stress pathways and how they contribute to the stress tolerance of tomato, one of the important vegetable crops, cultivated under varied environmental conditions.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Estrés del Retículo Endoplásmico , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Empalme del ARN , Conformación de Ácido Nucleico , ARN Mensajero
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