RESUMEN
Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.
Asunto(s)
Sitios de Empalme de ARN , Empalme del ARN , Humanos , Sitios de Empalme de ARN/genética , Intrones/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.
Asunto(s)
Hipoxia de la Célula , Motas Nucleares , Empalme del ARN , Factores de Empalme Serina-Arginina , Humanos , Empalme Alternativo , Exones/genética , Fosfoproteínas/genética , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Células HeLaRESUMEN
Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA) MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.
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Neoplasias de la Mama/genética , Neoplasias Pulmonares/genética , Proteínas de Unión al ARN/genética , Hipoxia Tumoral/genética , Factor A de Crecimiento Endotelial Vascular/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Transcriptoma/genéticaRESUMEN
BACKGROUND: Long non-coding RNAs (lncRNAs) represent a novel class of non-coding RNAs having a crucial role in many biological processes. The identification of long non-coding homologs among different species is essential to investigate such roles in model organisms as homologous genes tend to retain similar molecular and biological functions. Alignment-based metrics are able to effectively capture the conservation of transcribed coding sequences and then the homology of protein coding genes. However, unlike protein coding genes the poor sequence conservation of long non-coding genes makes the identification of their homologs a challenging task. RESULTS: In this study we compare alignment-based and alignment-free string similarity metrics and look at promoter regions as a possible source of conserved information. We show that promoter regions encode relevant information for the conservation of long non-coding genes across species and that such information is better captured by alignment-free metrics. We perform a genome wide test of this hypothesis in human, mouse, and zebrafish. CONCLUSIONS: The obtained results persuaded us to postulate the new hypothesis that, unlike protein coding genes, long non-coding genes tend to preserve their regulatory machinery rather than their transcribed sequence. All datasets, scripts, and the prediction tools adopted in this study are available at https://github.com/bioinformatics-sannio/lncrna-homologs .
Asunto(s)
Secuencia Conservada , Regulación de la Expresión Génica , Genoma , ARN Largo no Codificante/genética , Alineación de Secuencia/métodos , Animales , Humanos , Ratones , Pez Cebra/genéticaRESUMEN
Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.
Asunto(s)
Elementos de Facilitación Genéticos , Genómica , Animales , Ratones , Genómica/métodos , Mapeo Cromosómico/métodos , Regiones Promotoras Genéticas , EncéfaloRESUMEN
BACKGROUND: Management of spinal muscular atrophy (SMA) has progressed enormously and reached unprecedented levels with nusinersen gene therapy. We are finally able to counter the progression of this devastating genetic disease, contributing to the definition of new trajectories in its natural history and the identification of new SMA phenotypes post-gene therapy. The aim of this paper was to use the The International Classification of Functioning, Disability and Health-Children and Youth as a framework for the management of spinal muscular atrophy in the era of gene therapy: a proof-of-concept study (ICF-CY) as a comprehensive documentation tool to better understand and improve care provided to a child with SMA and to illustrate its use in a multidisciplinary perspective with a proof-of-concept study. CASE REPORT: An SMA child under gene therapy receiving a rehabilitation program. Clinical and functional outcome measures assessed at all levels of the ICF-CY, including impairment by Hammersmith Infant Neurological Examination, activity by Hammersmith Functional Motor Scale and Functional Independence Measure for Children, and participation by Pediatric Quality of Life Inventory™ - PedsQL™ and Neuromuscular Module™ as well as by parent report. Treatment outcomes were assessed at two main time points: at T0: prior to administration of nusinersen, and T1: immediately before the first administration of maintenance doses, 6 months after the first administration of nusinersen. A significant clinical improvement was seen on all domains between T0 and T1. The patient improved especially in motor skills and motor disability severity. The HRQOL showed a substantial improvement, too. ICF-CY codes were used to document change in body functions or structures, performance of activities or participation in social roles both in terms of gradient and hierarchy of change. This proof-of-concept study is the first attempt to explore SMA in a comprehensive manner from the perspective of the ICF-CY using a selected set of codes. These codes define essential child dimensions that can make up an ICF-CY core set, as identified by a trained multidisciplinary team, to guide assessment, treatment and rehabilitation. CLINICAL REHABILITATION IMPACT: Although limited to a single patient, this study makes nonetheless a strong point: we suggest using the ICF-CY as an essential tool in SMA management at a time when gene therapy with nusinersen is changing the phenotypes of activity and functioning in these children.
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Terapia Genética , Clasificación Internacional del Funcionamiento, de la Discapacidad y de la Salud , Atrofia Muscular Espinal/clasificación , Atrofia Muscular Espinal/terapia , Evaluación de la Discapacidad , Humanos , Lactante , Masculino , Atrofia Muscular Espinal/fisiopatología , Modalidades de Fisioterapia , Prueba de Estudio ConceptualRESUMEN
Hypoxia is associated with several diseases, including cancer. Cells that are deprived of adequate oxygen supply trigger transcriptional and post-transcriptional responses, which control cellular pathways such as angiogenesis, proliferation, and metabolic adaptation. Circular RNAs (circRNAs) are a novel class of mainly non-coding RNAs, which have been implicated in multiple cancers and attract increasing attention as potential biomarkers. Here, we characterize the circRNA signatures of three different cancer cell lines from cervical (HeLa), breast (MCF-7), and lung (A549) cancer under hypoxia. In order to reliably detect circRNAs, we integrate available tools with custom approaches for quantification and statistical analysis. Using this consolidated computational pipeline, we identify ~12000 circRNAs in the three cancer cell lines. Their molecular characteristics point to an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, such as the RNA-binding protein HNRNPC. Notably, we detect a number of circRNAs that are more abundant than their linear counterparts. In addition, 64 circRNAs significantly change in abundance upon hypoxia, in most cases in a cell type-specific manner. In summary, we present a comparative circRNA profiling in human cancer cell lines, which promises novel insights into the biogenesis and function of circRNAs under hypoxic stress.
Asunto(s)
Hipoxia de la Célula/fisiología , ARN Circular/genética , Células A549 , Hipoxia de la Célula/genética , Línea Celular Tumoral , Biología Computacional , Exones/genética , Células HeLa , Humanos , Intrones/genética , Células MCF-7 , MicroARNs/genética , RNA-SeqRESUMEN
Cerebral palsy (CP) is one of the most frequent causes of child disability in developed countries. Children with CP need lifelong assistance and care. The current prevalence of CP in industrialized countries ranges from 1.5 to 2.5 per 1,000 live births, with one new case every 500 live births. Children with CP have an almost normal life expectancy and mortality is very low. Despite the low mortality rate, 5%-10% of them die during childhood, especially when the severe motor disability is comorbid with epilepsy and severe intellectual disability. Given this life expectancy, children with CP present with a lifelong disability of varying severity and complexity, which requires individualized pathways of care. There are no specific treatments that can remediate the brain damage responsible for the complex clinical-functional dysfunctions typical of CP. There are, however, a number of interventions (eg, neurorehabilitation, functional orthopedic surgery, medication, etc) aimed at limiting the damage secondary to the brain insult and improving these patients' activity level and participation and, therefore, their quality of life. The extreme variability of clinical aspects and the complexity of affected functions determine a multifaceted skill development in children with CP. There is a need to provide them with long-term care, taking into account medical and social aspects as well as rehabilitation, education, and assistance. This long-term care must be suited according to children's developmental stage and their physical, psychological, and social development within their life contexts. This impacts heavily on the national health systems which must set up a network of services for children with CP, and it also impacts heavily on the family as a whole, due to the resulting distress, adjustment efforts, and changes in quality of life. This contribution is a narrative review of the current literature on long-term care for children with CP, aiming at suggesting reflections to improve these children's care.