Asunto(s)
Empalme del ARN , Ribosa , ARN de Planta/genética , Biología , ARN/genética , ARN/metabolismo , Precursores del ARNRESUMEN
The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1).
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , MicroARNs/biosíntesis , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Luciferasas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transgenes/genéticaRESUMEN
The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Plantas Modificadas Genéticamente , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Small RNA (sRNA)-mediated gene silencing constitutes a powerful tool for the molecular characterization- of a given gene. The RNAi technology has been largely used for this purpose. This approach is based on the cloning of an inverted repeated fragment of the gene to be silenced. Even when this approach -produces a strong repression of the target gene it also involves the production of multiple small RNAs species that can easily lead to off targeting. Taking advantage of the latest insights into the new post-biogenesis layer of regulation in microRNA (miRNA) activity, it is possible to overcome the above-mentioned limitation. Artificial microRNAs (amiRNAs) are 21mer small RNAs, which can be genetically engineered and they function to specifically silence single or multiple genes of interest. Since generally just one miRNA molecule is generated from each precursor, the specificity of this technology is much higher than longer inverted repeats. Application of this technology results in highly specific mRNA downregulation by computationally designed sequences programmed to target one or a set of custom-selected transcripts.
Asunto(s)
Ingeniería Genética/métodos , MicroARNs/genética , Interferencia de ARN , Arabidopsis/genética , Bryopsida/genética , Chlamydomonas reinhardtii/genética , Clonación Molecular , Oryza/genéticaRESUMEN
Small RNA suppression constitutes one of the major difficulties for a full molecular characterization of their specific roles in plants. Taking advantage of the latest insights into the new post-biogenesis layer of regulation in microRNA (miRNA) activity, it is possible to overcome the above-mentioned limitation (Nat Genet 39:1033-1037, 2007). We engineered the IPS1 non-coding RNA to bear a complementary sequence to a given miRNA family, resulting in specific sequestration of RISC complexes. MIMIC technology allows for the constitutive release of all of the potential targets of a miRNA family as well as tissue-specific and inducible suppression of its activity.
