RESUMO
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).
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , MicroRNAs/biossíntese , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Primers do DNA/genética , Luciferases , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transgenes/genéticaRESUMO
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.