RESUMO
Sertraline, a selective serotonin reuptake inhibitor (SSRI), is commonly used to treat various psychiatric disorders such as depression and anxiety due to its ability to increase serotonin availability in the brain. Recent findings suggest that sertraline may also influence the expression of genes related to synaptic plasticity and neuronal signaling pathways. Alternative splicing, a process that allows a single gene to produce multiple protein isoforms, plays a crucial role in the regulation of neuronal functions and plasticity. Dysregulation of alternative splicing events has been linked to various neurodevelopmental and neurodegenerative diseases. This study aims to explore the effects of sertraline on alternative splicing events, including exon inclusion, exon exclusion, and mutually exclusive splicing events, in genes associated with neuronal function in Drosophila melanogaster and to use this model to investigate the molecular impacts of SSRIs on gene regulation in the nervous system. RNA sequencing (RNA-seq) was performed on central nervous system samples from Drosophila melanogaster adults exposed to sertraline for 24 h when they were third instar larvae. Alternative splicing events were analyzed to identify changes in exon inclusion and exclusion, as well as intron retention. Sertraline treatment significantly altered alternative splicing patterns in key genes related to neuronal stability and function. Specifically, sertraline promoted the inclusion of long Ank2 isoforms, suggesting enhanced axonal stability, and favored long ATPalpha isoforms, which support Na+/K+ ATPase activity essential for ionic balance and neuronal excitability. Intron retention in the yuri gene suggests that cytoskeletal reorganization could impact neuronal morphology. Additionally, splicing alterations in sxc and Atg18a indicate a potential influence of sertraline on epigenetic regulation and autophagy processes, fundamental aspects for neuronal plasticity and cellular homeostasis. These findings suggest that sertraline influences alternative splicing in the central nervous system of Drosophila melanogaster, potentially contributing to its therapeutic effects by modulating neuronal stability and adaptability.
Assuntos
Processamento Alternativo , Proteínas de Drosophila , Drosophila melanogaster , Larva , Sertralina , Animais , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Processamento Alternativo/efeitos dos fármacos , Sertralina/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Éxons/genética , Tecido Nervoso/efeitos dos fármacos , Tecido Nervoso/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
In Arabidopsis thaliana, the transcription factors WRKY7, WRKY11 and WRKY17 act as negative defence regulators against Pseudomonas syringae pv. tomato (Pst) DC3000. However, their coordinated regulation of gene expression has yet to be fully explored. In this study, we conducted a transcriptomic analysis on the triple mutant wrky7/11/17 in response to Pst DC3000 at 0, 3 and 24 h post-inoculation (hpi). Our results suggest that at early infection stages (0 and 3 hpi), WRKY7, WRKY11 and WRKY17 significantly repress a group of genes involved in signal perception and transduction, including receptor-like kinases. Furthermore, at later stages of interaction (24 hpi), these transcription factors induce genes related to the biosynthesis and signalling of the jasmonic acid (JA) pathway. Further infection experiments with Pst DC3000 in plants treated with methyl jasmonate (a JA analogue) and infections with Botrytis cinerea, a pathogen against which JA-mediated responses are crucial for effective defence, support this proposal. Moreover, we analysed the role of WRKY7, WRKY11 and WRKY17 in alternative splicing regulation. A comparison between differentially expressed (DEG) and spliced (DAS) genes revealed that over 80% of DAS events do not occur in conjunction with overall changes in gene expression. Alternative splicing events were found in genes with functions in splicing and the JA pathway, such as ALY4, PRP40A, JAZ3 and JAZ10. These results suggest that WRKY7, WRKY11 and WRKY17 can also participate in this layer of gene expression regulation to modulate immunity negatively.
Assuntos
Processamento Alternativo , Proteínas de Arabidopsis , Arabidopsis , Botrytis , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Oxilipinas , Doenças das Plantas , Pseudomonas syringae , Fatores de Transcrição , Pseudomonas syringae/patogenicidade , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Arabidopsis/genética , Arabidopsis/microbiologia , Arabidopsis/imunologia , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Processamento Alternativo/genética , Botrytis/patogenicidade , Transdução de Sinais/genética , Acetatos/farmacologia , Redes Reguladoras de GenesRESUMO
The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.
Assuntos
Processamento Alternativo , Isoformas de Proteínas , Proteína Supressora de Tumor p53 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Humanos , Animais , Evolução Molecular , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Regulação da Expressão GênicaRESUMO
Mammalian Ste-20-like Kinases 1 and 2 (MST1/2) are core serine-threonine kinases of the Hippo pathway regulating several cellular processes, including cell cycle arrest and cell death. Here, we discovered a novel alternative splicing variant of the MST2 encoding gene, STK3, in malignant cells and tumor datasets. This variant, named STK3∆7 or MST2∆7 (for mRNA or protein, respectively), resulted from the skipping of exon 7. MST2∆7 exhibited increased ubiquitylation and interaction with the E3 ubiquitin-protein ligase CHIP compared to the full-length protein (MST2FL). Exon 7 in STK3 encodes a segment within the kinase domain, and its exclusion compromised MST2 interaction with and phosphorylation of MOB, a major MST1/2 substrate. Nevertheless, MST2∆7 was capable of interacting with MST1 and MST2FL. Unlike MST2FL, overexpression of MST2∆7 did not lead to increased cell death and growth arrest. Strikingly, we observed the exclusion of STK3 exon 7 in 3.2-15% of tumor samples from patients of several types of cancer, while STK3∆7 was seldomly found in healthy tissues. Our study identified a novel STK3 splicing variant with loss of function and the potential to disturb tissue homeostasis by impacting on MST2 activities in the regulation of cell death and quiescence.
Assuntos
Processamento Alternativo , Proliferação de Células , Proteínas Serina-Treonina Quinases , Serina-Treonina Quinase 3 , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Éxons/genética , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinase 3/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.
Assuntos
Arabidopsis , Cloroplastos , Regulação da Expressão Gênica de Plantas , Luz , Poliadenilação , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cloroplastos/genética , Processamento Alternativo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoAssuntos
Melanoma , Osteopontina , Humanos , Melanoma/genética , Melanoma/diagnóstico , Osteopontina/genética , Osteopontina/metabolismo , Prognóstico , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Processamento Alternativo , Feminino , MasculinoRESUMO
Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.
Assuntos
Neoplasias da Mama , Proteína Semelhante a ELAV 1 , Glutaminase , Glutaminase/metabolismo , Glutaminase/genética , Glutaminase/antagonistas & inibidores , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regulação Neoplásica da Expressão Gênica , Processamento Alternativo , Proliferação de Células , Glutamina/metabolismo , Estabilidade de RNARESUMO
RNA processing is a highly conserved mechanism that serves as a pivotal regulator of gene expression. Alternative processing generates transcripts that can still be translated but lead to potentially nonfunctional proteins. A plethora of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategically manipulate the host's RNA processing machinery to circumvent antiviral responses. We integrated publicly available omics datasets to systematically analyze isoform-level expression and delineate the nascent peptide landscape of SARS-CoV-2-infected human cells. Our findings explore a suggested but uncharacterized mechanism, whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling, interferon-stimulated (ISGs), class I MHC, and splicing machinery genes, including IRF7, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6 and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs an unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.
Assuntos
Processamento Alternativo , COVID-19 , Interferons , Isoformas de Proteínas , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/virologia , COVID-19/genética , COVID-19/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferons/metabolismo , Interferons/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismoRESUMO
Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.
Assuntos
Encéfalo , Distrofina , Regulação da Expressão Gênica no Desenvolvimento , Isoformas de Proteínas , Animais , Distrofina/genética , Distrofina/metabolismo , Encéfalo/metabolismo , Encéfalo/crescimento & desenvolvimento , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Camundongos , Camundongos Endogâmicos C57BL , Processamento Alternativo/genética , Ratos , Masculino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Animais Recém-NascidosRESUMO
Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat, and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance.
Assuntos
Processamento Alternativo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Animais , Humanos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Evolução Molecular , Isoformas de Proteínas/genética , Camundongos , Neoplasias/genética , RatosRESUMO
Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.
Assuntos
Processamento Alternativo , Arthrodermataceae , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Tinha , Fatores de Transcrição , Humanos , Arthrodermataceae/genética , Arthrodermataceae/metabolismo , Glucose/metabolismo , Queratinócitos/microbiologia , Queratinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tinha/metabolismo , Tinha/microbiologiaRESUMO
Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress.
Assuntos
Processamento Alternativo , Proteínas de Arabidopsis , Arabidopsis , Arginina , Regulação da Expressão Gênica de Plantas , Proteína-Arginina N-Metiltransferases , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Processamento Alternativo/genética , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Estresse Fisiológico/genética , Arginina/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Mutação/genéticaRESUMO
Targeted therapy indications for actionable variants in non-small-cell lung cancer (NSCLC) have primarily been studied in Caucasian populations, with limited data on Latin American patients. This study utilized a 52-genes next-generation sequencing (NGS) panel to analyze 1560 tumor biopsies from NSCLC patients in Chile, Brazil, and Peru. The RNA sequencing reads and DNA coverage were correlated to improve the detection of the actionable MET exon 14 skipping variant (METex14). The pathogenicity of MET variants of uncertain significance (VUSs) was assessed using bioinformatic methods, based on their predicted driver potential. The effects of the predicted drivers VUS T992I and H1094Y on c-MET signaling activation, proliferation, and migration were evaluated in HEK293T, BEAS-2B, and H1993 cell lines. Subsequently, c-Met inhibitors were tested in 2D and 3D cell cultures, and drug affinity was determined using 3D structure simulations. The prevalence of MET variants in the South American cohort was 8%, and RNA-based diagnosis detected 27% more cases of METex14 than DNA-based methods. Notably, 20% of METex14 cases with RNA reads below the detection threshold were confirmed using DNA analysis. The novel actionable T992I and H1094Y variants induced proliferation and migration through c-Met/Akt signaling. Both variants showed sensitivity to crizotinib and savolitinib, but the H1094Y variant exhibited reduced sensitivity to capmatinib. These findings highlight the importance of RNA-based METex14 diagnosis and reveal the drug sensitivity profiles of novel actionable MET variants from an understudied patient population.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Éxons , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas c-met , Humanos , Proteínas Proto-Oncogênicas c-met/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/diagnóstico , Éxons/genética , Linhagem Celular Tumoral , Masculino , Feminino , Células HEK293 , Mutação , Processamento Alternativo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Crizotinibe/uso terapêutico , Crizotinibe/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Pessoa de Meia-Idade , América Latina/epidemiologiaRESUMO
Aluminum (Al) toxicity limits crop production worldwide. Although studies have identified genes associated with Al tolerance in crops, a large amount of data remains unexplored using other strategies. Here, we searched for single substitutions and InDels across differentially expressed genes (DEGs), linked DEGs to Al-tolerance QTLs reported in the literature for common maize, and investigated the alternative splicing regulated by Al3+ toxicity. We found 929 substitutions between DEGs in Al-tolerant and 464 in Al-sensitive inbred lines, of which 165 and 80 were non-synonymous, respectively. Only 12 NS variants had deleterious predicted effect on protein function in Al-tolerant and 13 in Al-sensitive. Moreover, 378 DEGs were mapped in Al-QTL regions for the Al-tolerant and 213 for the Al-sensitive. Furthermore, Al stress is primarily regulated at the transcriptional level in popcorn. Important genes identified, such as HDT1, SWEET4a, GSTs, SAD9, PIP2-2, CASP-like 5, and AGP, may benefit molecular assisted popcorn breeding or be useful in biotechnological approaches. These findings offer insights into the mechanisms of Al tolerance in popcorn and provide a 'hypothesis-free' strategy for identifying and prioritizing candidate genes that could be used to develop molecular markers or cultivars resilient to acidic soils.
Assuntos
Alumínio , Transcriptoma , Alumínio/toxicidade , Zea mays/genética , Produtos Agrícolas , Processamento AlternativoRESUMO
Piscirickettsia salmonis, an intracellular bacterium in salmon aquaculture, is a big challenge because it is responsible for 54.2% of Atlantic salmon mortalities. In recent years, the high relevance of Alternative Splicing (AS) as a molecular mechanism associated with infectious conditions and host-pathogen interaction processes, especially in host immune activation, has been observed. Several studies have highlighted the role of AS in the host's immune response during viral, bacterial, and endoparasite infection. In the present study, we evaluated AS transcriptome profiles during P. salmonis infection in the two most used study models, SHK-1 cell line and salmon head kidney tissue. First, the SHK-1 cell line was exposed to P. salmonis infection at 0-, 7-, and 14-days post-infection (dpi). Following, total RNA was extracted for Illumina sequencing. On the other hand, RNA-Seq datasets of Atlantic salmon head kidney infected with the same P. salmonis strayingwase used. For both study models, the highest number of differentially alternative splicing (DAS) events was observed at 7 dpi, 16,830 DAS events derived from 9213 DAS genes in SHK-1 cells, and 13,820 DAS events from 7684 DAS genes in salmon HK. Alternative first exon (AF) was the most abundant AS type in the three infection times analyzed, representing 31% in SHK-1 cells and 228.6 in salmon HK; meanwhile, mutually exclusive exon (MX) was the least abundant. Notably, functional annotation of DAS genes in SHK-1 cells infected with P. salmonis showed a high presence of genes related to nucleotide metabolism. In contrast, the salmon head kidney exhibited many GO terms associated with immune response. Our findings reported the role of AS during P. salmonis infection in Atlantic salmon. These studies would contribute to a better understanding of the molecular bases that support the pathogen-host interaction, evidencing the contribution of AS regulating the transcriptional host response.
Assuntos
Doenças dos Peixes , Piscirickettsia , Infecções por Piscirickettsiaceae , Salmo salar , Animais , Transcriptoma , Salmo salar/genética , Rim Cefálico , Processamento Alternativo , Piscirickettsia/fisiologia , Linhagem Celular , Infecções por Piscirickettsiaceae/genética , Infecções por Piscirickettsiaceae/veterináriaRESUMO
Light is both the main source of energy and a key environmental signal for plants. It regulates not only gene expression but also the tightly related processes of splicing and alternative splicing (AS). Two main pathways have been proposed to link light sensing with the splicing machinery. One occurs through a photosynthesis-related signal, and the other is mediated by photosensory proteins, such as red light-sensing phytochromes. Here, we evaluated the relative contribution of each of these pathways by performing a transcriptome-wide analysis of light regulation of AS in plants that do not express any functional phytochrome (phyQ). We found that an acute 2-h red-light pulse in the middle of the night induces changes in the splicing patterns of 483 genes in wild-type plants. Approximately 30% of these genes also showed strong light regulation of splicing patterns in phyQ mutant plants, revealing that phytochromes are important but not essential for the regulation of AS by R light. We then performed a meta-analysis of related transcriptomic datasets and found that different light regulatory pathways can have overlapping targets in terms of AS regulation. All the evidence suggests that AS is regulated simultaneously by various light signaling pathways, and the relative contribution of each pathway is highly dependent on the plant developmental stage.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/genética , Arabidopsis/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Processamento Alternativo/genética , Splicing de RNA , Plantas/metabolismoRESUMO
Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function.
Assuntos
Etanol , Transtornos do Espectro Alcoólico Fetal , Gravidez , Feminino , Camundongos , Humanos , Animais , Etanol/toxicidade , RNA , Processamento Alternativo , Transtornos do Espectro Alcoólico Fetal/genética , Biologia ComputacionalRESUMO
BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.
Assuntos
Metilação de DNA , Linho , Linho/genética , Secas , Processamento Alternativo/genética , Estresse Fisiológico/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , TranscriptomaRESUMO
The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.
Assuntos
Proteínas Serina-Treonina Quinases , Splicing de RNA , Processamento Alternativo , Fatores de Processamento de RNA/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Processamento U2AF/química , Fatores de Transcrição/metabolismo , Transição Epitelial-Mesenquimal , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Sea lice are pathogenic marine ectoparasite copepods that represent a severe risk to the worldwide salmon industry. Several transcriptomic investigations have characterized the regulation of gene expression response of Atlantic salmon to sea lice infestation. These studies have focused on the levels of transcript, overlooking the potentially relevant role of alternative splicing (AS), which corresponds to an essential control mechanism of gene expression through RNA processing. In the present study, we performed a genome-wide bioinformatics characterization of differential AS event dynamics in control and infested C. rogercresseyi Atlantic salmon and in resistant and susceptible phenotypes. We identified a significant rise of alternative splicing events and AS genes after infestation and 176 differential alternative splicing events (DASE) from 133 genes. In addition, a higher number of DASE and AS genes were observed among resistant and susceptible phenotypes. Functional annotation of AS genes shows several terms and pathways associated with behavior, RNA splicing, immune response, and RNA binding. Furthermore, three protein-coding genes were identified undergoing differential transcript usage events, among resistant and susceptible phenotypes. Our findings support AS performing a relevant regulatory role in the response of salmonids to sea lice infestation.