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1.
Science ; 373(6551): 231-236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244417

RESUMO

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células-Tronco/enzimologia , Células-Tronco/virologia , Processamento Alternativo , Animais , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , RNA Helicases DEAD-box/química , Humanos , Imunidade Inata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Organoides/enzimologia , Organoides/virologia , Infecções por Vírus de RNA/enzimologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/enzimologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
2.
BMC Bioinformatics ; 22(1): 368, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34266387

RESUMO

BACKGROUND: Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. RESULTS: Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns' overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. CONCLUSIONS: Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q.


Assuntos
Processamento Alternativo , Sítios de Splice de RNA , Genoma , Humanos , Íntrons , Sítios de Splice de RNA/genética , Splicing de RNA/genética
3.
Nat Commun ; 12(1): 4230, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244494

RESUMO

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXß2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXß2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXß2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXß2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.


Assuntos
Processamento Alternativo , Carcinoma Epitelial do Ovário/genética , Proteínas da Matriz Extracelular/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Ovarianas/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ovário/patologia , Ovário/cirurgia , Fosforilação/genética , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Commun ; 12(1): 4203, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244519

RESUMO

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.


Assuntos
Processamento Alternativo , Cardiomiopatia Dilatada/genética , Miócitos Cardíacos/patologia , Proteínas de Ligação a RNA/genética , RNA-Seq/métodos , Sistemas CRISPR-Cas/genética , Cardiomiopatia Dilatada/patologia , Diferenciação Celular/genética , Linhagem Celular , Estudos de Viabilidade , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Proteínas Musculares/genética , Mutação , Isoformas de RNA/genética , RNA Guia/genética
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(6): 507-512, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34060445

RESUMO

Objective To investigate the interaction between Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and its biological functions. Methods The interaction between WASH/FAM21, the core member of WASH complex, and hnRNP A1 was identified by mass spectrometry and co-immunoprecipitation. Telomere lengths of shWASH and shScramble cells were measured by Real-time qPCR. RNA-seq was used to generate the transcription profiles of shWASH and shScramble cells. Results Mass spectrometry results indicated that a variety of hnRNP family members, including hnRNP A1, interact with FAM21-d219N. The interaction between endogenous WASH/FAM21 and hnRNP A1 was confirmed by co-immunoprecipitation results. Depletion of WASH did not affect the relative length of telomeres, but it significantly increased the exon skipping and led to the variation of transcriptional expression of hundreds of genes, including some NF-κB target genes. Conclusion WASH in the nucleus interacts with hnRNP A1 to participate in the regulation of alternative splicing and gene transcription.


Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Transcrição Genética
6.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067970

RESUMO

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.


Assuntos
Citoesqueleto de Actina/metabolismo , Processamento Alternativo , Citoplasma/metabolismo , Éxons , Tropomiosina/metabolismo , Humanos , Isoformas de Proteínas , Tropomiosina/química , Tropomiosina/genética
7.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068052

RESUMO

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.


Assuntos
Processamento Alternativo , Clonagem Molecular , Genes Reporter , Doenças Genéticas Inatas/diagnóstico , Vetores Genéticos/genética , Genoma Humano , Mutação , Enzimas de Restrição do DNA , Doenças Genéticas Inatas/genética , Humanos
8.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070203

RESUMO

Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.


Assuntos
Regiões 3' não Traduzidas , Biomarcadores Tumorais/genética , Processamento Alternativo , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Poliadenilação , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Análise de Célula Única
9.
World J Gastroenterol ; 27(21): 2871-2894, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34135559

RESUMO

BACKGROUND: Alternative splicing (AS) increases the diversity of mRNA during transcription; it might play a role in alteration of the immune microenvironment, which could influence the development of immunotherapeutic strategies against cancer. AIM: To obtain the transcriptomic and clinical features and AS events in stomach adenocarcinoma (STAD) from the database. The overall survival data associated with AS events were used to construct a signature prognostic model for STAD. METHODS: Differentially expressed immune-related genes were identified between subtypes on the basis of the prognostic model. In STAD, 2042 overall-survival-related AS events were significantly enriched in various pathways and influenced several cellular functions. Furthermore, the network of splicing factors and overall-survival-associated AS events indicated potential regulatory mechanisms underlying the AS events in STAD. RESULTS: An eleven-AS-signature prognostic model (CD44|14986|ES, PPHLN1|21214|AT, RASSF4|11351|ES, KIAA1147|82046|AP, PPP2R5D|76200|ES, LOH12CR1|20507|ES, CDKN3|27569|AP, UBA52|48486|AD, CADPS|65499|AT, SRSF7| 53276|RI, and WEE1|14328|AP) was constructed and significantly related to STAD overall survival, immune cells, and cancer-related pathways. The differentially expressed immune-related genes between the high- and low-risk score groups were significantly enriched in cancer-related pathways. CONCLUSION: This study provided an AS-related prognostic model, potential mechanisms for AS, and alterations in the immune microenvironment (immune cells, genes, and pathways) for future research in STAD.


Assuntos
Adenocarcinoma , Processamento Alternativo , Adenocarcinoma/genética , Antígenos de Neoplasias , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Nucleares , Proteína Fosfatase 2 , Estômago , Microambiente Tumoral
10.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071104

RESUMO

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.


Assuntos
Colina O-Acetiltransferase/biossíntese , Neurônios Colinérgicos/metabolismo , Gânglios Espinais/citologia , Proteínas do Tecido Nervoso/biossíntese , Células Receptoras Sensoriais/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/biossíntese , Acetilcolina/metabolismo , Processamento Alternativo , Animais , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurogênese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/citologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
11.
Molecules ; 26(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064652

RESUMO

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.


Assuntos
Processamento Alternativo/genética , Proteína Semelhante a ELAV 4/genética , Neocórtex/metabolismo , Poliadenilação/genética , Animais , Proteína Semelhante a ELAV 4/metabolismo , Éxons/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Gene ; 794: 145752, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34082065

RESUMO

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general.


Assuntos
Envelhecimento/genética , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/efeitos dos fármacos , Glucuronidase/genética , Fígado/química , Compostos Fitoquímicos/administração & dosagem , Envelhecimento/efeitos dos fármacos , Processamento Alternativo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Resposta ao Choque Térmico , Íntrons , Japão , Fígado/efeitos dos fármacos , Medicina Tradicional , Metabolômica , Camundongos , Modelos Animais , Compostos Fitoquímicos/farmacologia , Precursores de RNA/genética , Análise de Sequência de RNA
13.
BMC Plant Biol ; 21(1): 280, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34154536

RESUMO

Alternative splicing (AS) increases the diversity of transcripts and proteins through the selection of different splice sites and plays an important role in the growth, development and stress tolerance of plants. With the release of the reference genome of the tea plant (Camellia sinensis) and the development of transcriptome sequencing, researchers have reported the existence of AS in tea plants. However, there is a lack of a platform, centered on different RNA-seq datasets, that provides comprehensive information on AS.To facilitate access to information on AS and reveal the molecular function of AS in tea plants, we established the first comprehensive AS database for tea plants (TeaAS, http://www.teaas.cn/index.php ). In this study, 3.96 Tb reads from 66 different RNA-seq datasets were collected to identify AS events. TeaAS supports four methods of retrieval of AS information based on gene ID, gene name, annotation (non-redundant/Kyoto encyclopedia of genes and genomes/gene ontology annotation or chromosomal location) and RNA-seq data. It integrates data pertaining to genome annotation, type of AS event, transcript sequence, and isoforms expression levels from 66 RNA-seq datasets. The AS events resulting from different environmental conditions and that occurring in varied tissue types, and the expression levels of specific transcripts can be clearly identified through this online database. Moreover, it also provides two useful tools, Basic Local Alignment Search Tool and Generic Genome Browser, for sequence alignment and visualization of gene structure.The features of the TeaAS database make it a comprehensive AS bioinformatics platform for researchers, as well as a reference for studying AS events in woody crops. It could also be helpful for revealing the novel biological functions of AS in gene regulation in tea plants.


Assuntos
Processamento Alternativo , Camellia sinensis/genética , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , RNA de Plantas , RNA-Seq
14.
BMC Bioinformatics ; 22(1): 347, 2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174808

RESUMO

BACKGROUND: Computational tools analyzing RNA-sequencing data have boosted alternative splicing research by identifying and assessing differentially spliced genes. However, common alternative splicing analysis tools differ substantially in their statistical analyses and general performance. This report compares the computational performance (CPU utilization and RAM usage) of three event-level splicing tools; rMATS, MISO, and SUPPA2. Additionally, concordance between tool outputs was investigated. RESULTS: Log-linear relations were found between job times and dataset size in all splicing tools and all virtual machine (VM) configurations. MISO had the highest job times for all analyses, irrespective of VM size, while MISO analyses also exceeded maximum CPU utilization on all VM sizes. rMATS and SUPPA2 load averages were relatively low in both size and replicate comparisons, not nearing maximum CPU utilization in the VM simulating the lowest computational power (D2 VM). RAM usage in rMATS and SUPPA2 did not exceed 20% of maximum RAM in both size and replicate comparisons while MISO reached maximum RAM usage in D2 VM analyses for input size. Correlation coefficients of differential splicing analyses showed high correlation (ß > 80%) between different tool outputs with the exception of comparisons of retained intron (RI) events between rMATS/MISO and rMATS/SUPPA2 (ß < 60%). CONCLUSIONS: Prior to RNA-seq analyses, users should consider job time, amount of replicates and splice event type of interest to determine the optimal alternative splicing tool. In general, rMATS is superior to both MISO and SUPPA2 in computational performance. Analysis outputs show high concordance between tools, with the exception of RI events.


Assuntos
Laboratórios , Software , Processamento Alternativo , Splicing de RNA , Análise de Sequência de RNA
15.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069457

RESUMO

The nuclear thyroid hormone receptors (THRs) are key mediators of thyroid hormone function on the cellular level via modulation of gene expression. Two different genes encode THRs (THRA and THRB), and are pleiotropically involved in development, metabolism, and growth. The THRA1 and THRA2 isoforms, which result from alternative splicing of THRA, differ in their C-terminal ligand-binding domain (LBD). Most published disease-associated THRA variants are located in the LBD of THRA1 and impede triiodothyronine (T3) binding. This keeps the nuclear receptor in an inactive state and inhibits target gene expression. Here, we investigated a new dominant THRA variant (chr17:g.38,241,010A > G, GRCh37.13 | c.518A > G, NM_199334 | p.(E173G), NP_955366), which is located between the DNA- and ligand-binding domains and affects both splicing isoforms. Patients presented partially with hypothyroid (intellectual disability, motor developmental delay, brain atrophy, and constipation) and partially with hyperthyroid symptoms (tachycardia and behavioral abnormalities) to varying degrees. Functional characterization of THRA1p.(E173G) by reporter gene assays revealed increased transcriptional activity in contrast to THRA1(WT), unexpectedly revealing the first gain-of-function mutation found in THRA1. The THRA2 isoform does not bind T3 and antagonizes THRA1 action. Introduction of p.(E173G) into THRA2 increased its inhibitory effect on THRA1, which helps to explain the hypothyroid symptoms seen in our patients. We used protein structure models to investigate possible underlying pathomechanisms of this variant with a gain-of-antagonistic function and suggest that the p.(E173G) variant may have an influence on the dimerization domain of the nuclear receptor.


Assuntos
Genes erbA/genética , Receptores dos Hormônios Tireóideos/metabolismo , Doenças da Glândula Tireoide/genética , Adulto , Processamento Alternativo/genética , Família , Feminino , Mutação com Ganho de Função/genética , Expressão Gênica/genética , Genes erbA/fisiologia , Humanos , Hipotireoidismo/metabolismo , Mutação/genética , Linhagem , Isoformas de Proteínas/metabolismo , Receptores dos Hormônios Tireóideos/genética , Irmãos , Glândula Tireoide/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo
16.
Methods Enzymol ; 654: 345-364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120721

RESUMO

Alternative splicing of RNA transcripts allows a single gene to generate multiple products and is a key means of generating functionally diverse voltage-gated ion channels. Splicing can be regulated according to cell type, cell state, and stage of development to produce a bespoke complement of protein isoforms. Characterizing the identities of full-length transcript isoforms is essential in order to fully understand a gene's expression and function. However, the repertoire of transcript isoforms is not well characterized for most genes. Long read nanopore sequencing allows full-length isoforms to be sequenced, therefore identifying full-length transcripts. Using this approach, we recently discovered that the human CACNA1C gene gives rise to a far greater repertoire of splice isoforms than previously appreciated. Here we provide a detailed overview of the technical approach we used to achieve this. The method described in this chapter combines long read nanopore sequencing with PCR targeting to selectively sequence transcripts of a specific gene of interest.


Assuntos
Processamento Alternativo , Splicing de RNA , Perfilação da Expressão Gênica , Humanos , Canais Iônicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
17.
J Cell Sci ; 134(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34137440

RESUMO

Hypoxia is a severe stressor to cellular homeostasis. At the cellular level, low oxygen triggers the transcription of a variety of genes supporting cell survival and oxygen homeostasis mediated by transcription factors, such as hypoxia-inducible factors (HIFs). Among many determinants dictating cell responses to hypoxia and HIFs are microRNAs (miRNAs). Cajal bodies (CBs), subnuclear structures involved in ribonucleoprotein biogenesis, have been recently proven to contribute to miRNA processing and biogenesis but have not been studied under hypoxia. Here, we show, for the first time, a hypoxia-dependent increase in CB number in WI-38 primary fibroblasts, which normally have very few CBs. Additionally, the CB marker protein coilin is upregulated in hypoxic WI-38 cells. However, the hypoxic coilin upregulation was not seen in transformed cell lines. Furthermore, we found that coilin is needed for the hypoxic induction of a well-known hypoxia-induced miRNA (hypoxamiR), miR-210, as well as for the hypoxia-induced alternative splicing of the miR-210 host gene, MIR210HG. These findings provide a new link in the physiological understanding of coilin, CBs and miRNA dysregulation in hypoxic pathology.


Assuntos
MicroRNAs , Processamento Alternativo/genética , Corpos Enovelados/genética , Corpos Enovelados/metabolismo , Humanos , Hipóxia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo
18.
Nucleic Acids Res ; 49(11): 6213-6237, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086943

RESUMO

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.


Assuntos
Processamento Alternativo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Éxons , Feminino , Células HeLa , Código das Histonas , Humanos , Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
19.
Nucleic Acids Res ; 49(11): 6165-6180, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107020

RESUMO

The current understanding of how overall principles of translational control govern the embryo-to-adult transition in mammals is still far from comprehensive. Herein we profiled the translatomes and transcriptomes of six tissues from the mice at embryonic and adult stages and presented the first report of tissue- and stage-specific translational landscape in mice. We quantified the extent of gene expression divergence among different expression layers, tissues and stages, detected significant changes in gene composition and function underlying these divergences and revealed the changing architecture of translational regulation. We further showed that dynamic translational regulation can be largely achieved via modulation of translational efficiency. Translational efficiency could be altered by alternative splicing (AS), upstream and downstream open reading frames (uORFs and dORFs). We revealed AS-mediated translational repression that was exerted in an event type-dependent manner. uORFs and dORFs exhibited mutually exclusive usage and the opposing effects of translational regulation. Furthermore, we discovered many novel microproteins encoded by long noncoding RNAs and demonstrated their regulatory potential and functional relevance. Our data and analyses will facilitate a better understanding of the complexity of translation and translational regulation across tissue and stage spectra and provide an important resource to the translatome research community.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Processamento Alternativo , Animais , Embrião de Mamíferos/metabolismo , Camundongos Endogâmicos C57BL , Fases de Leitura Aberta , Especificidade de Órgãos , RNA Longo não Codificante/metabolismo , RNA-Seq , Transcriptoma
20.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073089

RESUMO

Sox proteins are known as crucial transcription factors for many developmental processes and for a wide range of common diseases. They were believed to specifically bind and bend DNA with other transcription factors and elicit transcriptional activation or repression activities in the early stage of transcription. However, their functions are not limited to transcription initiation. It has been showed that Sox proteins are involved in the regulation of alternative splicing regulatory networks and translational control. In this review, we discuss the current knowledge on how Sox transcription factors such as Sox2, Sry, Sox6, and Sox9 allow the coordination of co-transcriptional splicing and also the mechanism of SOX4-mediated translational control in the context of RNA polymerase III.


Assuntos
Fatores de Transcrição SOX , Processamento Alternativo , Animais , Humanos , Biossíntese de Proteínas , RNA Polimerase III , Splicing de RNA , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/fisiologia , Spliceossomos/metabolismo , Ativação Transcricional
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