RESUMO
OBJECTIVE: Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA- (miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype. METHODS: After preliminary microarrays screening, miR-29b levels were measured by RT- quantitative PCR in in vitro models of chondrocyte phenotype changes (IL-1ß challenge or serial subculturing) and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identified in silico in 3'-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining) during chondrogenesis. RESULTS: MiR-29b level increased significantly in IL-1ß-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3'-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased. CONCLUSIONS: By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.
Assuntos
Cartilagem Articular/metabolismo , Desdiferenciação Celular , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Regiões 3' não Traduzidas , Cartilagem Articular/patologia , Condrócitos/patologia , Feminino , Humanos , Masculino , Osteoartrite/patologiaRESUMO
Stem-loop qRT-PCR is one of the most commonly used real-time PCR approach to quantify small non-coding RNAs such as microRNAs. The quantification method is divided in two steps. First, RNA is reverse-transcribed using a specific stem-loop primer, and the resulting RT product is subsequently used as a template for quantitative real-time PCR. This fast and simple method provides quantitative data with high sensitivity and specificity to study miRNAs and their functions.
Assuntos
Pequeno RNA não Traduzido/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Primers do DNA/genética , Sequências Repetidas Invertidas/genética , Pequeno RNA não Traduzido/genéticaRESUMO
This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression.
Assuntos
Inativação Gênica/fisiologia , Genes Reporter/genética , Luciferases/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Regiões não Traduzidas/genética , Clonagem Molecular , Células HeLa , Humanos , Luciferases/genética , Medições Luminescentes/métodos , RNA Mensageiro/genéticaRESUMO
Small nucleolar RNAs or snoRNAs, principally implicated in post-transcriptional chemical modification of other RNAs, were among the first non-coding RNA identified, together with ribosomal and transfer RNA. Lately, snoRNA have been involved in various unexpected functions, which renewed researcher's interest for these molecules. SnoRNA processing into smaller functional RNA species (sdRNA for snoRNA-derived RNA) or into miRNA (sno-miR), snoRNA mediated regulation of messenger RNA alternative splicing or snoRNA links to human disorders, including cancers, are some of the topics developed in this review.
Assuntos
RNA Nucleolar Pequeno/fisiologia , Animais , Enzimas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transporte Proteico/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
After transcription, mRNA editing in angiosperm chloroplasts and mitochondria results in the conversion of cytidine to uridine by deamination. Analysis of Arabidopsis thaliana mutants affected in RNA editing have shown that many pentatricopeptide repeat proteins (PPRs) are required for specific cytidine deamination events. PPR proteins have been shown to be sequence-specific RNA binding proteins allowing the recognition of the C to be edited. The C-terminal DYW domain present in many editing factors has been proposed to catalyze C deamination, as it shows sequence similarities with cytidine deaminases in other organisms. However, many editing factors, such as the first to be discovered, CHLORORESPIRATORY REDUCTION4 (CRR4), lack this domain, so its importance has been unclear. Using a reverse genetic approach, we identified DYW1, an RNA editing factor acting specifically on the plastid ndhD-1 editing site recognized by CRR4. Unlike other known editing factors, DYW1 contains no identifiable PPR motifs but does contain a clear DYW domain. We were able to show interaction between CRR4 and DYW1 by bimolecular fluorescence complementation and to reconstitute a functional chimeric CRR4-DYW1 protein complementing the crr4 dyw1double mutant. We propose that CRR4 and DYW1 act together to edit the ndhD-1 site.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Edição de RNA/genética , RNA de Plantas/genética , Alelos , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Mutação , Plastídeos/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de SequênciaRESUMO
RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.
Assuntos
Modelos Biológicos , Organelas/enzimologia , Organelas/genética , Plantas/enzimologia , Plantas/genética , Edição de RNA/genética , Sequência de Aminoácidos , Sítios de Ligação , Citidina Desaminase/química , Citidina Desaminase/metabolismo , Bases de Dados de Proteínas , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de ProteínaRESUMO
Oxa1p is a key component of the machinery for the insertion of membrane proteins in mitochondria, and in the yeast Saccharomyces cerevisiae, the deletion of OXA1 impairs the biogenesis of the three respiratory complexes of dual genetic origin. Oxa1p is formed from three domains located in the intermembrane space, the inner membrane and the mitochondrial matrix. We have isolated a high copy suppressor able to partially compensate for the respiratory deficiency caused by a large deletion of the matrix domain. We show that the suppressor gene corresponds to the nuclear transcriptional activator Hap4p which is known to regulate respiratory functions.
