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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445352

RESUMO

The Masculinizer (Masc) gene has been known to control sex development and dosage compensation in lepidopterans. However, it remains unclear whether its ortholog exists and plays the same roles in distantly related lepidopterans such as Helicoverpa armigera. To address this question, we cloned Masc from H. armigera (HaMasc), which contains all essential functional domains of BmMasc, albeit with less than 30% amino acid sequence identity with BmMasc. Genomic PCR and qPCR analyses showed that HaMasc is a Z chromosome-linked gene since its genomic content in males (ZZ) was two times greater than that in females (ZW). RT-PCR and RT-qPCR analyses revealed that HaMasc expression was sex- and stage-biased, with significantly more transcripts in males and eggs than in females and other stages. Transfection of a mixture of three siRNAs of HaMasc into a male embryonic cell line of H. armigera led to the appearance of female-specific mRNA splicing isoforms of H. armigeradoublesex (Hadsx), a downstream target gene of HaMasc in the H. armigera sex determination pathway. The knockdown of HaMasc, starting from the third instar larvae resulted in a shift of Hadsx splicing from male to female isoforms, smaller male pupa and testes, fewer but larger/longer spermatocytes and sperm bundles, delayed pupation and internal fusion of the testes and follicles. These data demonstrate that HaMasc functions as a masculinizing gene in the H. armigera sex-determination cascade.


Assuntos
Proteínas de Insetos/fisiologia , Mariposas/genética , Processos de Determinação Sexual/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Compensação de Dosagem (Genética) , Feminino , Proteínas de Insetos/genética , Larva , Masculino , Mariposas/classificação , Filogenia , Isoformas de RNA , Análise de Sequência de DNA , Cromossomos Sexuais
2.
Biochim Biophys Acta Gene Regul Mech ; 1864(10): 194746, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34419630

RESUMO

Lamin A/C proteins, major components of the nuclear lamina, are encoded by the LMNA gene. These proteins have multiple cellular functions, including DNA transcription and replication, chromatin organization, regulation of the cell cycle, and apoptosis. Mutations in LMNA are associated with a variety of diseases called laminopathies. LMNA has implications in cancer; however, its mechanisms of dysregulation in cancer cells are not yet fully understood. In this study, among the LMNA transcript variants, we focused on a transcriptional variant 6 (termed LMNA-V6), which contains unique 3 exons upstream of exon 1 of LMNA. The promoter region of LMNA-V6 formed multiple G-quadruplexes and increased its transcriptional activity. Moreover, LMNA-V6 negatively regulated other LMNA mRNA variants, lamin A and lamin C, via direct interaction with their promoter. Knockdown of LMNA-V6 decreased the proliferation of colon cancer cells, whereas overexpression of the unique 3 exons of LMNA-V6 increased cell growth. Furthermore, microarray gene expression profiling showed that alteration of LMNA-V6 levels influenced the expression of p53 in colon cancer cells. Taken together, the results suggest that LMNA-V6 may be a novel functional RNA whose expression is regulated through multiple G-quadruplexes in colon cancer cells.


Assuntos
Neoplasias do Colo/genética , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Lamina Tipo A/genética , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Humanos , Lamina Tipo A/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Splicing de RNA , Transcrição Genética
3.
Nat Commun ; 12(1): 4825, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376658

RESUMO

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.


Assuntos
Encéfalo/metabolismo , Éxons/genética , Íntrons/genética , Sequenciamento por Nanoporos/métodos , RNA Circular/genética , Análise de Sequência de RNA/métodos , Animais , Expressão Gênica , Humanos , Masculino , Camundongos da Linhagem 129 , Isoformas de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Nat Commun ; 12(1): 4872, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381052

RESUMO

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.


Assuntos
Apoptose , Vasos Sanguíneos/crescimento & desenvolvimento , Receptores de Netrina/metabolismo , Isoformas de RNA/metabolismo , Processamento Alternativo , Animais , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Células Endoteliais , Humanos , Morfogênese , Neovascularização Patológica/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina/genética , Netrina-1/metabolismo , Isoformas de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Peixe-Zebra
5.
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
6.
Cancer Invest ; 39(6-7): 559-570, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043476

RESUMO

The study evaluated whether SPP1/osteopontin (OPN) splice variants are differentially expressed in nonmelanoma skin cancer compared to normal skin. The absolute number of mRNA molecules of OPN-a predominated in normal skin and nonmelanoma skin cancer compared to OPN-b, OPN-c, and OPN-5. However, mRNAs of OPN-a, OPN-b, and OPN-c were expressed in higher levels in cutaneous squamous cell carcinomas (cSCCs) and basal cell carcinomas relative to normal skin. Additionally, OPN-5 expression was higher than OPN-b and OPN-c, and OPN-c, in normal skin and nonmelanoma skin cancer, respectively. Furthermore, we identified four OPN-5 splice variants, which were cloned and analyzed for protein expression.


Assuntos
Processamento Alternativo , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Osteopontina/metabolismo , Neoplasias Cutâneas/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Feminino , Regulação Neoplásica da Expressão Gênica , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Osteopontina/genética , Isoformas de RNA/metabolismo , Neoplasias Cutâneas/metabolismo , Regulação para Cima
7.
Biochem Biophys Res Commun ; 560: 152-158, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989907

RESUMO

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.


Assuntos
Processamento Alternativo , Distrofina/genética , Células-Tronco Neurais/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Distrofina/metabolismo , Células-Tronco Neurais/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de RNA/metabolismo , Ratos Wistar
8.
Brief Bioinform ; 22(5)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33855356

RESUMO

MicroRNA (miRNA) is not a single sequence, but a series of multiple variants (also termed isomiRs) with sequence and expression heterogeneity. Whether and how these isoforms contribute to functional variation and complexity at the systems and network levels remain largely unknown. To explore this question systematically, we comprehensively analyzed the expression of small RNAs and their target sites to interrogate functional variations between novel isomiRs and their canonical miRNA sequences. Our analyses of the pan-cancer landscape of miRNA expression indicate that multiple isomiRs generated from the same miRNA locus often exhibit remarkable variation in their sequence, expression and function. We interrogated abundant and differentially expressed 5' isomiRs with novel seed sequences via seed shifting and identified many potential novel targets of these 5' isomiRs that would expand interaction capabilities between small RNAs and mRNAs, rewiring regulatory networks and increasing signaling circuit complexity. Further analyses revealed that some miRNA loci might generate diverse dominant isomiRs that often involved isomiRs with varied seeds and arm-switching, suggesting a selective advantage of multiple isomiRs in regulating gene expression. Finally, experimental validation indicated that isomiRs with shifted seed sequences could regulate novel target mRNAs and therefore contribute to regulatory network rewiring. Our analysis uncovers a widespread expansion of isomiR and mRNA interaction networks compared with those seen in canonical small RNA analysis; this expansion suggests global gene regulation network perturbations by alternative small RNA variants or isoforms. Taken together, the variations in isomiRs that occur during miRNA processing and maturation are likely to play a far more complex and plastic role in gene regulation than previously anticipated.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias/genética , Isoformas de RNA/genética , Análise por Conglomerados , Redes Reguladoras de Genes , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/genética , Análise de Sobrevida
9.
Nat Commun ; 12(1): 2014, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795677

RESUMO

Age-associated changes in gene expression in skeletal muscle of healthy individuals reflect accumulation of damage and compensatory adaptations to preserve tissue integrity. To characterize these changes, RNA was extracted and sequenced from muscle biopsies collected from 53 healthy individuals (22-83 years old) of the GESTALT study of the National Institute on Aging-NIH. Expression levels of 57,205 protein-coding and non-coding RNAs were studied as a function of aging by linear and negative binomial regression models. From both models, 1134 RNAs changed significantly with age. The most differentially abundant mRNAs encoded proteins implicated in several age-related processes, including cellular senescence, insulin signaling, and myogenesis. Specific mRNA isoforms that changed significantly with age in skeletal muscle were enriched for proteins involved in oxidative phosphorylation and adipogenesis. Our study establishes a detailed framework of the global transcriptome and mRNA isoforms that govern muscle damage and homeostasis with age.


Assuntos
Envelhecimento Saudável/genética , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Homeostase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Musculares/genética , Isoformas de RNA/genética , RNA não Traduzido/genética , Adulto Jovem
10.
Methods Mol Biol ; 2284: 231-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33835446

RESUMO

High-throughput sequencing for micro-RNAs (miRNAs) to obtain expression estimates is a central method of molecular biology. Surprisingly, there are a number of different approaches to converting sequencing output into micro-RNA counts. Each has their own strengths and biases that impact on the final data that can be obtained from a sequencing run. This chapter serves to make the reader aware of the trade-offs one must consider in analyzing small RNA sequencing data. It then compares two methods, miRge2.0 and the sRNAbench and the steps utilized to output data from their tools.


Assuntos
Biologia Computacional/métodos , MicroRNAs/genética , Análise de Sequência de RNA/métodos , Animais , Simulação por Computador , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/análise , Polimorfismo de Nucleotídeo Único , Isoformas de RNA/análise , Isoformas de RNA/genética , Software
11.
Nat Biotechnol ; 39(7): 836-845, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33707777

RESUMO

Reconstructing the sequence of circular RNAs (circRNAs) from short RNA sequencing reads has proved challenging given the similarity of circRNAs and their corresponding linear messenger RNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length circRNAs. Here we describe a protocol for enrichment and full-length sequencing of circRNA isoforms using nanopore technology. Circular reverse transcription and size selection achieves a 20-fold higher enrichment of circRNAs from total RNA compared to previous methods. We developed an algorithm, called circRNA identifier using long-read sequencing data (CIRI-long), to reconstruct the sequence of circRNAs. The workflow was validated with simulated data and by comparison to Illumina sequencing as well as quantitative real-time RT-PCR. We used CIRI-long to analyze adult mouse brain samples and systematically profile circRNAs, including mitochondria-derived and transcriptional read-through circRNAs. We identified a new type of intronic self-ligated circRNA that exhibits special splicing and expression patterns. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length circRNA sequences.


Assuntos
Sequenciamento por Nanoporos , RNA Circular/genética , Animais , Sequência de Bases , Simulação por Computador , Regulação da Expressão Gênica , Humanos , Camundongos , Isoformas de RNA , Reprodutibilidade dos Testes
12.
J Cell Sci ; 134(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33674450

RESUMO

The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3' untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.


Assuntos
Axônios , Isoformas de RNA , Axônios/metabolismo , Lipoilação , Regeneração Nervosa , Isoformas de RNA/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
13.
PLoS Genet ; 17(3): e1009263, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33684133

RESUMO

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3' UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.


Assuntos
Regulação da Expressão Gênica , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno/genética , RNA Mensageiro/genética , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Poliadenilação , Isoformas de RNA , Transporte de RNA , Transcrição Genética , Transcriptoma
14.
PLoS Genet ; 17(3): e1009402, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33739979

RESUMO

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo , Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/metabolismo , Morfogênese/genética , Alelos , Animais , Animais Geneticamente Modificados , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ordem dos Genes , Testes Genéticos , Variação Genética , Fígado/metabolismo , Modelos Biológicos , Mutação , Fenótipo , Isoformas de RNA , Peixe-Zebra , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
15.
Development ; 148(4)2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33531432

RESUMO

KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.


Assuntos
Diferenciação Celular/genética , Movimento Celular/genética , Regulação da Expressão Gênica , Cinesina/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Processamento Alternativo , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Perfilação da Expressão Gênica , Cinesina/metabolismo , Camundongos , Mutação , Neurogênese/genética , Proteômica/métodos , Isoformas de RNA , Proteínas Repressoras/metabolismo
16.
Aging (Albany NY) ; 13(4): 5136-5149, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33535175

RESUMO

A close association between peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) and the development of diabetic retinopathy (DR) has been previously suggested. Herein, a meta-analysis was conducted to explore the association between PPAR-γ2 polymorphisms and DR risk by performing a systematic search and quantitative analysis. Overall, fourteen articles involving 10,527 subjects were included. The pooled results did not reveal an association between PPAR-γ2 rs1801282 C/G and DR susceptibility in the overall population (e.g., the dominant model: CG+GG vs. CC, OR=0.85, 95% CI=0.69-1.06, P=0.15, I2=62.9%). Furthermore, heterogeneity tests, cumulative analyses, sensitivity analyses, and publication bias analyses were conducted and showed that the results were robust. Similarly, race-based subgroup analyses and other subgroup analyses did not reveal an association between the rs1801282 C/G and DR susceptibility. In addition, no significant association was observed between PPAR-γ2 rs3856806 C/T polymorphism and DR risk (e.g., the dominant model: CT+TT vs. CC, OR=1.12, 95%CI=0.91-1.37, P=0.28, I2=27.0%). Overall, based on the current sample size and the level of evidence presented in the study, the results suggest that PPAR-γ2 gene polymorphisms are not associated with DR risk.


Assuntos
Retinopatia Diabética/genética , PPAR gama/genética , Processamento Alternativo , Predisposição Genética para Doença , Humanos , Polimorfismo de Nucleotídeo Único , Isoformas de RNA
17.
Mol Neurobiol ; 58(5): 2046-2060, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33411244

RESUMO

The Shaker-related potassium channel Kv1.1 subunit has important implications for controlling neuronal excitabilities. A particular recoding by A-to-I RNA editing at I400 of Kv1.1 mRNA is an underestimated mechanism for fine-tuning the properties of Kv1.1-containing channels. Knowledge about functional differences between edited (I400V) and non-edited Kv1.1 isoforms is insufficient, especially in neurons. To understand their different roles, the two Kv1.1 isoforms were overexpressed in the prefrontal cortex via local adeno-associated virus-mediated gene delivery. The I400V isoform showed a higher competitiveness in membrane translocalization, but failed to reduce current-evoked discharges and showed weaker impact on spiking-frequency adaptation in the transduced neurons. The non-edited Kv1.1 overexpression led to slight elevations in both fast- and non-inactivating current components of macroscopic potassium current. By contrast, the I400V overexpression did not impact the fast-inactivating current component. Further isolation of Kv1.1-specific current by its specific blocker dendrotoxin-κ showed that both isoforms did result in significant increases in current amplitude, whereas the I400V was less efficient in contributing the fast-inactivating current component. Voltage-dependent properties of the fast-inactivating current component did not alter for both isoforms. For recovery kinetics, the I400V showed a significant acceleration of recovery from fast inactivation. The gene delivery of the I400V rather than the wild type exhibited anxiolytic activities, which was assessed by an open field test. These results suggest that the Kv1.1 RNA editing isoforms have different properties and outcomes, reflecting the functional and phenotypic significance of the Kv1.1 RNA editing in neurons.


Assuntos
Canal de Potássio Kv1.1/metabolismo , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Canal de Potássio Kv1.1/genética , Memória de Curto Prazo/fisiologia , Camundongos , Atividade Motora/fisiologia , Edição de RNA , Isoformas de RNA
18.
Nat Commun ; 12(1): 520, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483506

RESUMO

The fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of acute myeloid leukemia. Here we show that the RUNX1/RUNX1T1 oncogene is a regulator of alternative RNA splicing in leukemic cells. The comprehensive analysis of RUNX1/RUNX1T1-associated splicing events identifies two principal mechanisms that underlie the differential production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start site selection, and (ii) direct or indirect control of the expression of genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5'-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals alternative splicing as an important component of transcriptome re-organization in leukemia by an aberrant transcriptional regulator.


Assuntos
Processamento Alternativo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Doença Aguda , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Mieloide/patologia , Modelos Genéticos , Proteínas de Fusão Oncogênica/metabolismo , Interferência de RNA , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Sítio de Iniciação de Transcrição
19.
Nat Commun ; 12(1): 266, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436621

RESUMO

Circular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. Short-read RNA sequencing (RNA-seq) is a widely used strategy to identify circRNAs. However, an inherent limitation of short-read RNA-seq is that it does not experimentally determine the full-length sequences and exact exonic compositions of circRNAs. Here, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification followed by nanopore long-read sequencing. We describe an integrated computational pipeline to reliably characterize full-length circRNA isoforms using isoCirc data. Using isoCirc, we generate a comprehensive catalog of 107,147 full-length circRNA isoforms across 12 human tissues and one human cell line (HEK293), including 40,628 isoforms ≥500 nt in length. We identify widespread alternative splicing events within the internal part of circRNAs, including 720 retained intron events corresponding to a class of exon-intron circRNAs (EIciRNAs). Collectively, isoCirc and the companion dataset provide a useful strategy and resource for studying circRNAs in human transcriptomes.


Assuntos
Bases de Dados Genéticas , Isoformas de RNA/genética , RNA Circular/genética , Transcriptoma/genética , Processamento Alternativo/genética , Células HEK293 , Humanos , RNA-Seq
20.
Cell Mol Life Sci ; 78(5): 2213-2230, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32914209

RESUMO

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Oligonucleotídeos Antissenso/genética , Poli A/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Estudos de Coortes , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poli A/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo
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