RESUMO
MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , MicroRNAs/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serrate-Jagged , Nicotiana/metabolismoRESUMO
A method was established to isolate and quantify small amounts of chitin-binding mistletoe lectin (cbML) from extracts of the mistletoe (Viscum album L.) by affinity and reverse phase high performance liquid chromatography. A validation, according to ICH guidelines, of this analytical method was carried out and showed that specificity, robustness and precision are guaranteed. In addition, linearity is ensured for a content between 0.6 and 4.1 microg/ml of cbML in the extracts and recovery was calculated to be in the range of 94 to 100%. So, accuracy of the method is guaranteed as well. As far as the range of the analytical method is concerned, a minimum of 1.2 microg and a maximum of 8.2 microg cbML can be incubated with the affinity material. Detection and quantitation limits were calculated to be 0.13 and 0.46 microg/ml cbML, respectively.
