RESUMEN
Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Integrasas/fisiología , Intrones/fisiología , Neoplasias/fisiopatología , Roturas del ADN de Doble Cadena , Dependovirus , Recombinación Homóloga , Humanos , Neoplasias/enzimología , Neoplasias/genéticaRESUMEN
PURPOSE: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. EXPERIMENTAL DESIGN: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. RESULTS: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. CONCLUSIONS: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.
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Neoplasias Encefálicas/genética , ADN-Topoisomerasas de Tipo II/fisiología , Epigénesis Genética/fisiología , Glioma/genética , Intrones/fisiología , Oncogenes/fisiología , Proteínas de Unión a Poli-ADP-Ribosa/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Neoplasias Encefálicas/enzimología , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Humanos , RatonesRESUMEN
AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Glutamina/metabolismo , Neuronas Motoras/patología , Demencia Frontotemporal/genética , Glutamina/genética , Humanos , Intrones/fisiología , Prolina/genética , Prolina/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
OBJECTIVE: Stroke is a cerebrovascular disorder that often causes neurological function defects. ARPP21 is a conserved host gene of miR-128 controlling neurodevelopmental functions. This study investigated the mechanism of ARPP21 antagonistic intron miR-128 on neurological function repair after stroke. METHODS: Expressions of ARPP21 and miR-128 in stroke patients were detected. The mouse neurons and astrocytes were cultured in vitro and treated with oxygen-glucose deprivation (OGD). The OGD-treated cells were transfected with pc-ARPP21 and miR-128 mimic. The proliferation of astrocytes, and the apoptosis of neurons and astrocytes were detected, and inflammatory factors of astrocytes were measured. The binding relationship between miR-128 and CREB1 was verified. The rat model of middle cerebral artery occlusion (MCAO) was established. ARPP21 expression in model rats was detected. The effects of pc-ARPP21 on neuron injury, brain edema volume, and cerebral infarct in rats were observed. RESULTS: ARPP21 expression was downregulated and miR-128 expression was upregulated in stroke patients. pc-ARPP21 facilitated the proliferation of astrocytes and inhibited apoptosis of neurons and astrocytes, and reduced inflammation of astrocytes. miR-128 mimic could reverse these effects of pc-ARPP21 on neurons and astrocytes. miR-128 targeted CREB1 and reduced BDNF secretion. In vitro experiments confirmed that ARPP21 expression was decreased in MCAO rats, and pc-ARPP21 promoted neurological function repair after stroke. CONCLUSION: ARPP21 upregulated CREB1 and BDNF expressions by antagonizing miR-128, thus inhibiting neuronal apoptosis and promoting neurological function repair after stroke. This study may offer a novel target for the management of stroke.
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Isquemia Encefálica/metabolismo , Intrones/fisiología , MicroARNs/biosíntesis , Fosfoproteínas/biosíntesis , Accidente Cerebrovascular/metabolismo , Adulto , Anciano , Animales , Isquemia Encefálica/patología , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/patologíaRESUMEN
Essential genes have been studied by copy number variants and deletions, both associated with introns. The premise of our work is that introns of essential genes have distinct characteristic properties. We provide support for this by training a deep learning model and demonstrating that introns alone can be used to classify essentiality. The model, limited to first introns, performs at an increased level, implicating first introns in essentiality. We identify unique properties of introns of essential genes, finding that their structure protects against deletion and intron-loss events, especially centered on the first intron. We show that GC density is increased in the first introns of essential genes, allowing for increased enhancer activity, protection against deletions, and improved splice site recognition. We find that first introns of essential genes are of remarkably smaller size than their nonessential counterparts, and to protect against common 3' end deletion events, essential genes carry an increased number of (smaller) introns. To demonstrate the importance of the seven features we identified, we train a feature-based model using only these features and achieve high performance.
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Genes Esenciales/genética , Intrones/genética , Empalme Alternativo/genética , Secuencia de Bases/genética , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN/genética , Bases de Datos Genéticas , Aprendizaje Profundo , Exones/genética , Genes Esenciales/fisiología , Humanos , Mutación INDEL/genética , Intrones/fisiologíaRESUMEN
BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.
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Evolución Biológica , ADN Protozoario/análisis , Dinoflagelados/citología , Dinoflagelados/genética , Orgánulos/fisiología , Proteínas Protozoarias/análisis , Secuencia de Bases , Evolución Molecular , Intrones/fisiologíaRESUMEN
We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.
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Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Proteínas de Unión al ARN/fisiología , Cromosoma X/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Intrones/genética , Intrones/fisiología , Masculino , Proteínas de Unión al ARN/metabolismo , Procesos de Determinación del SexoRESUMEN
Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csß1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class.
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Basidiomycota/genética , Regulación Fúngica de la Expresión Génica , Intrones/genética , Polyporales/genética , Moléculas de Adhesión Celular/genética , Proteínas Fúngicas/genética , Intrones/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Regiones Promotoras Genéticas , Proteínas RecombinantesRESUMEN
The expression of AtSUC1 is controlled by the promoter and intragenic sequences. AtSUC1 is expressed in roots, pollen and trichomes. However, AtSUC1 promoter-GUS transgenics only show expression in trichomes and pollen. Here, we show that the root expression of AtSUC1 is controlled by an interaction between the AtSUC1 promoter and two short introns. The deletion of either intron from whole-gene-GUS constructs results in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. In Arabidopsis seedlings, exogenously applied sucrose induces the expression of AtSUC1 in roots and causes anthocyanin accumulation. atsuc1 loss-of-function mutants are defective in sucrose-induced anthocyanin accumulation. We show that an AtSUC1 whole-gene-GUS construct expressing a nonfunctional AtSUC1 (D152N) mutant, that is transport inactive, is defective in sucrose-induced AtSUC1 expression when expressed in an atsuc1-null background. We also show that the transport-defective allele does not complement the loss of sucrose-induced anthocyanin accumulation in null atsuc1 mutants. The results indicate that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and sucrose-induced anthocyanin accumulation and that the site for sucrose detection is intracellular.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Intrones , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Intrones/fisiología , Proteínas de Transporte de Membrana/fisiología , Organismos Modificados Genéticamente , Proteínas de Plantas/fisiología , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas/fisiología , Plantones/metabolismo , Sacarosa/metabolismo , XenopusRESUMEN
The highly conserved heat shock protein 70 (HSP70) contributes to survival at a cellular level and greatly enhances stress tolerance in many organisms. In this study, we isolate and characterize Cshsp702, which encodes an inducible form of HSP70 in the rice stem borer, Chilo suppressalis. Cshsp702 does not contain introns; the translational product is comprised of 629 amino acids with an isoelectric point of 5.69. Real-time quantitative PCR revealed that Cshsp702 was expressed at maximal levels in hemocytes and was minimally expressed in the midgut. Expression of Cshsp702 in response to a range of temperatures (-11 to 43 °C) indicated significant induction by extreme cold and hot temperatures, with maximum expression after 2 h at 42 °C. The induction of Cshsp702 in response to the endoparasite Cotesia chilonis was also studied; interestingly, Cshsp702 expression in C. suppressalis was significantly induced at 24 h and 5 days, which correspond to predicted times of C. chilonis feeding and growth, respectively. The potential induction of Cshsp702 as an inflammatory response due to parasitic stress is discussed. In conclusion, Cshsp702 is induced in response to both environmental and biotic stress and plays an important role in the physiological adaptation of C. suppressalis.
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Ambiente , Proteínas HSP70 de Choque Térmico/metabolismo , Hemocitos/metabolismo , Estrés Fisiológico/genética , Secuencia de Aminoácidos/fisiología , Animales , Calor/efectos adversos , Intrones/fisiología , Larva/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are helical repeat RNA-binding proteins that function in RNA processing by conferring sequence-specific RNA-binding activity. Owing to the lethality of PPR mutants, functions of many PPR proteins remain obscure. In this study, we report the function of PPR20 in intron splicing in mitochondria and its role in maize seed development. PPR20 is a P-type PPR protein targeted to mitochondria. The ppr20 mutants display slow embryo and endosperm development. Null mutation of PPR20 severely reduces the cis-splicing of mitochondrial nad2 intron 3, resulting in reduction in the assembly and activity of mitochondrial complex I. The ppr20-35 allele with a Mu insertion in the N-terminal region shows a much weaker phenotype. Molecular analyses revealed that the mutant produces a truncated transcript, coding for PPR20ΔN120 lacking the N-terminal 120 amino acids. Subcellular localization revealed that PPR20ΔN120:GFP is able to target to mitochondria as well, suggesting the sequence diversity of the mitochondrial targeting peptides. Another mutant zm_mterf15 was also found to be impaired in the splicing of mitochondrial nad2 intron 3. Further analyses are required to identify the exact function of PPR20 and Zm_mTERF15 in the splicing of nad2 intron 3.
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Complejo I de Transporte de Electrón/metabolismo , Intrones/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Empalme del ARN , Semillas/crecimiento & desarrollo , Zea mays/crecimiento & desarrollo , Alelos , Complejo I de Transporte de Electrón/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Mitocondriales/genética , Mutación , Fenotipo , Desarrollo de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Unión al ARN , Semillas/citología , Semillas/genética , Zea mays/genéticaRESUMEN
BACKGROUND: Intronic (TTTCA)n insertions in the SAMD12, TNRC6A, and RAPGEF2 genes have been identified as causes of familial cortical myoclonic tremor with epilepsy. OBJECTIVE: To identify the cause of familial cortical myoclonic tremor with epilepsy pedigrees without (TTTCA)n insertions in SAMD12, TNRC6A, and RAPGEF2. METHODS: Repeat-primed polymerase chain reaction, long-range polymerase chain reaction, and Sanger sequencing were performed to identify the existence of a novel (TTTGA)n insertion. Targeted long-read sequencing was performed to confirm the accurate structure of the (TTTGA)n insertion. RESULTS: We identified a novel expanded intronic (TTTGA)n insertion at the same site as the previously reported (TTTCA)n insertion in SAMD12. This insertion cosegregated with familial cortical myoclonic tremor with epilepsy in 1 Chinese pedigree with no (TTTCA)n insertion. In the targeted long-read sequencing of 2 patients and 1 asymptomatic carrier in this pedigree, with 1 previously reported (TTTCA)n -insertion-carrying patient as a positive control, a respective total of 302, 159, 207, and 50 on-target subreads (predicated accuracy: ≥90%) spanning the target repeat expansion region were generated. These sequencing data revealed the accurate repeat expansion structures as (TTTTA)114-123 (TTTGA)108-116 in the pedigree and (TTTTA)38 (TTTCA)479 in (TTTCA)n -insertion-carrying patient. CONCLUSION: The targeted long-read sequencing helped us to elucidate the accurate structures of the (TTTGA)n and (TTTCA)n insertions. Our finding offers a novel possible cause for familial cortical myoclonic tremor with epilepsy and might shed light on the identification of genetic causes of this disease in pedigrees with no detected (TTTCA)n insertion in the reported causative genes. © 2019 International Parkinson and Movement Disorder Society.
Asunto(s)
Epilepsias Mioclónicas/genética , Proteínas del Tejido Nervioso/genética , Temblor/genética , Adulto , Pueblo Asiatico , Epilepsias Mioclónicas/complicaciones , Humanos , Intrones/fisiología , Masculino , Linaje , Temblor/complicacionesRESUMEN
Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (ß-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.
Asunto(s)
Evolución Biológica , Euglenozoos/clasificación , Biología Molecular , Trypanosomatina/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Euglénidos/clasificación , Euglénidos/genética , Euglenozoos/genética , Genoma/fisiología , Intrones/fisiología , Mitocondrias/genética , Procesos Fototróficos , Filogenia , Interferencia de ARN , ARN Ribosómico 28S/genética , Trypanosomatina/clasificación , Trypanosomatina/enzimologíaRESUMEN
Actinotrichia are the first exoskeletal elements formed during zebrafish fin development. These rigid fibrils serve as skeletal support for the fin fold and as substrates for mesenchymal cell migration. In the adult intact fins, actinotrichia are restricted to the distal domain of the fin. Following fin amputation, actinotrichia also reform during regeneration. The actinodin gene family codes for structural proteins of actinotrichia. We have previously identified cis-acting regulatory elements in a 2kb genomic region upstream of the first exon of actinodin1, termed 2P, required for tissue-specific expression in the fin fold ectoderm and mesenchyme during embryonic development. Indeed, 2P contains an ectodermal enhancer in a 150bp region named epi. Deletion of epi from 2P results in loss of ectodermal-specific activity. In the present study, we sought to further characterize the activity of these regulatory sequences throughout fin development and during adult fin regeneration. Using a reporter transgenic approach, we show that a site within the epi region, termed epi3, contains an early mesenchymal-specific repressor. We also show that the larval fin fold ectodermal enhancer within epi3 remains functional in the basal epithelial layer during fin regeneration. We show that the first non-coding exon and first intron of actinodin1 contains a transcriptional enhancer and an alternative promoter that are necessary for the persistence of reporter expression reminiscent of actinodin1 expression during adulthood. Altogether, we have identified cis-acting regulatory elements that are required for tissue-specific expression as well as full recapitulation of actinodin1 expression during adulthood. Furthermore, the characterization of these elements provides us with useful molecular tools for the enhancement of transgene expression in adulthood.
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Aletas de Animales/fisiología , Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regeneración/fisiología , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Embrión no Mamífero/citología , Elementos de Facilitación Genéticos/fisiología , Exones/fisiología , Intrones/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Pentatricopeptide repeat (PPR) proteins play crucial roles in intron splicing, which is important for RNA maturation. Identification of novel PPR protein with the function of intron splicing would help to understand the RNA splicing mechanism. In this study, we identified the maize empty pericarp602 (emp602) mutants, the mature kernels of which showed empty pericarp phenotype. We cloned the Emp602 gene from emp602 mutants and revealed that Emp602 encodes a mitochondrial-localized P-type PPR protein. We further revealed that Emp602 is specific for the cis-splicing of mitochondrial Nad4 intron 1 and intron 3, and mutation of Emp602 led to the loss of mature Nad4 transcripts. The loss of function of Emp602 nearly damaged the assembly and accumulation of complex I and arrested mitochondria formation, which arrested the seed development. The failed assembly of complex I triggers significant upregulation of Aox expression in emp602 mutants. Transcriptome analysis showed that the expression of mitochondrial-related genes, e.g. the genes associated with mitochondrial inner membrane presequence translocase complex and electron carrier activity, were extensively upregulated in emp602 mutant. These results demonstrate that EMP602 functions in the splicing of Nad4 intron 1 and intron 3, and the loss of function of Emp602 arrested maize seed development by disrupting the mitochondria complex I assembly.
Asunto(s)
Semillas/metabolismo , Zea mays/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Intrones/genética , Intrones/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Empalme del ARN/genética , Empalme del ARN/fisiología , Semillas/genética , Semillas/crecimiento & desarrollo , Zea mays/genética , Zea mays/crecimiento & desarrolloRESUMEN
MAIN CONCLUSION: Expression of the Galactinol synthase genes in rice is regulated through post-transcriptional intron retention in response to abiotic stress and may be linked to Raffinose Family Oligosaccharide synthesis in osmotic perturbation. Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the full-length pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.
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Galactosiltransferasas/metabolismo , Genes de Plantas/fisiología , Oryza/genética , Arabidopsis , Galactosiltransferasas/genética , Galactosiltransferasas/fisiología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Intrones/genética , Intrones/fisiología , Oryza/enzimología , Oryza/fisiología , Plantas Modificadas Genéticamente , Procesamiento Postranscripcional del ARN/genética , Procesamiento Postranscripcional del ARN/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Estrés FisiológicoAsunto(s)
Bacterias/genética , Intrones/genética , Empalme del ARN/genética , Evolución Biológica , Eucariontes/genética , Células Eucariotas/metabolismo , Células Eucariotas/fisiología , Evolución Molecular , Genoma , Intrones/fisiología , Células Procariotas/metabolismo , Células Procariotas/fisiología , ARN , Empalme del ARN/fisiología , ARN Mensajero/genética , Empalmosomas/genética , Empalmosomas/fisiologíaRESUMEN
The spliceosome is a multi-subunit RNA-protein complex involved in the removal of non-coding segments (introns) from between the coding regions (exons) in precursors of messenger RNAs (pre-mRNAs). Intron removal proceeds via two transesterification reactions, occurring between conserved sequences at intron-exon junctions. A tightly regulated, hierarchical assembly with a multitude of structural and compositional rearrangements posed a great challenge for structural studies of the spliceosome. Over the years, X-ray crystallography dominated the field, providing valuable high-resolution structural information that was mostly limited to individual proteins and smaller sub-complexes. Recent developments in the field of cryo-electron microscopy allowed the visualisation of fully assembled yeast and human spliceosomes, providing unprecedented insights into substrate recognition, catalysis, and active site formation. This has advanced our mechanistic understanding of pre-mRNA splicing enormously.
Asunto(s)
Precursores del ARN/metabolismo , Empalmosomas/metabolismo , Catálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Intrones/genética , Intrones/fisiología , Precursores del ARN/genética , Empalmosomas/genética , Empalmosomas/ultraestructuraRESUMEN
Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.