RESUMO
Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.
Assuntos
Processamento Alternativo/fisiologia , Éxons/fisiologia , Sítios de Splice de RNA/fisiologia , Sequência Consenso/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Células HeLa , Humanos , Íntrons , Precursores de RNA/fisiologia , Splicing de RNA/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Transcriptoma/genéticaRESUMO
Recursive splicing (RS) starts by defining an "RS-exon," which is then spliced to the preceding exon, thus creating a recursive 5' splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.
Assuntos
Processamento Alternativo/fisiologia , Éxons/fisiologia , Sítios de Splice de RNA/fisiologia , Animais , Linhagem Celular , Núcleo Celular , Drosophila , Células HEK293 , Células HeLa , Humanos , Íntrons , Células K562 , Camundongos , Proteínas Nucleares , Precursores de RNA/fisiologia , Splicing de RNA/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas/fisiologia , Transcriptoma/genéticaRESUMO
Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic ß-cell identity and function. Elimination of Lkb1 from the ß-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of ß cell-selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from ß-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors (TFs) important for ß-cell identity, such as FOXA, MAFA or RFX6, and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates ß-cell identity and function.
Assuntos
Epigênese Genética , Células Secretoras de Insulina , Proteínas Serina-Treonina Quinases , Animais , Células Secretoras de Insulina/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos Knockout , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Regiões Promotoras Genéticas , Camundongos Endogâmicos C57BL , MasculinoRESUMO
Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients' muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.
Assuntos
Distrofia Muscular do Cíngulo dos Membros , Humanos , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Masculino , Feminino , Adulto , Animais , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Linhagem , Drosophila melanogaster , Miofibrilas/patologia , Miofibrilas/genética , Miofibrilas/metabolismo , Pessoa de Meia-Idade , Fenótipo , Adolescente , Adulto Jovem , CriançaRESUMO
The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.
Assuntos
Genes cdc , Glioblastoma , Humanos , Splicing de RNA , Divisão Celular , Núcleo Celular/metabolismo , Glioblastoma/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies.
Assuntos
Biologia Computacional/métodos , Evolução Molecular , Perfilação da Expressão Gênica , Splicing de RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , HumanosRESUMO
It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.
Assuntos
Splicing de RNA/genética , Vertebrados/genética , Animais , Anquirinas/genética , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Códon de Terminação/genética , Drosophila melanogaster/genética , Éxons/genética , Feminino , Lobo Frontal/citologia , Lobo Frontal/metabolismo , Humanos , Imunoglobulinas/genética , Íntrons/genética , Masculino , Regiões Promotoras Genéticas/genética , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Sítios de Splice de RNA/genética , Estabilidade de RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
U1 snRNP (U1 small nuclear ribonucleoprotein) is an essential component of the splicing machinery. U1 snRNP also plays an additional role in 3'-end mRNA processing when it binds close to polyadenylation sites (PAS). Cotranscriptionally, U1 snRNP binding close to putative PAS prevents premature cleavage and polyadenylation and consequently safeguards pre-mRNA transcripts and defines promoter directionality. At the 3'-end of mRNAs, U1 snRNP binding to putative PAS may regulate mRNA length or inhibit polyadenylation and, therefore, gene expression. U1 interference (U1i) is a technique to inhibit gene expression based on the property of U1 snRNP to inhibit polyadenylation. It requires the expression of a modified U1 snRNP, which interacts with a target gene upstream of its PAS and inhibits target gene expression. U1i has been used to inhibit the expression of reporter or endogenous genes both in tissue culture and in animal models. In addition, U1i combination with RNA interference (RNAi), another RNA-based gene silencing technology, results in a synergistic increased inhibition. This is of special interest for antiviral therapy, where strong inhibitions may be required to decrease the expression of replicative viral RNAs and impact the replication cycle. Furthermore, the combination of U1i and RNAi-based inhibitors should prevent the appearance of viral variants resistant to the treatment and allows the dose of inhibitors to be decreased and a functional inhibition to be obtained with fewer off target effects. In fact, U1i has been used to inhibit the expression of HIV-1 and HBV, whose viral genomes express mRNAs that must be polyadenylated by the nuclear polyadenylation machinery. In the case of HBV, antiviral U1i has been combined with RNAi to demonstrate a strong inhibition of expression from HBV sequences in vivo. This shows that, although several aspects of U1i technology remain to be addressed, U1i and U1i combined with RNAi have great potential as antivirals.
Assuntos
Antivirais/uso terapêutico , Terapia de Alvo Molecular/métodos , Interferência de RNA , Ribonucleoproteína Nuclear Pequena U1/genética , Viroses/terapia , Animais , Humanos , Conformação Molecular , Interferência de RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Relação Estrutura-AtividadeRESUMO
Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.
Assuntos
Interferência de RNA , RNA Nuclear Pequeno/antagonistas & inibidores , Animais , Linhagem Celular , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic ß-cell identity and function. Elimination of Lkb1 from the ß-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of ß cell- selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from ß-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors important for ß-cell identity, such as FOXA, MAFA or RFX6 and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates ß-cell identity and function.
RESUMO
Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.
Assuntos
Éxons , Íntrons , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Oligonucleotídeos Antissenso/genética , RNA Nuclear Pequeno/genética , Processamento Alternativo , Calpaína/genética , Linhagem Celular , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Humanos , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Processamento de Serina-ArgininaRESUMO
Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.
Assuntos
Riboswitch , Animais , Camundongos , Riboswitch/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Tetraciclina/farmacologia , Antibacterianos , Mamíferos/genética , Expressão GênicaRESUMO
Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.
Assuntos
Cromatina , Origem de Replicação , Humanos , Animais , Complexo de Reconhecimento de Origem , Fosforilação , RNA , MamíferosRESUMO
Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.
RESUMO
INTRODUCTION: Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain-3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. METHODS: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. RESULTS: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain-3, suggesting "molecular complementation" in these patients. CONCLUSION: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain-3, resulting in an exceptionally benign phenotype.
Assuntos
Calpaína/genética , Triagem de Portadores Genéticos , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Índice de Gravidade de Doença , Adulto , Animais , Células COS , Chlorocebus aethiops , Feminino , Humanos , Masculino , Distrofia Muscular do Cíngulo dos Membros/diagnósticoRESUMO
During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.