Figure 5

Ath- MIR859-774 polycistronic pri- MIRNA is processed by a DCL1-dependent pathway. (a) Overexpression of Ath-MIR859-774 pri-MIRNA in dcl1.9 mutant and in the corresponding DCL1.9/dcl1.9 heterozygous siblings (three representative OverExpressing (OE) transgenic lines) is shown. Real-time RT-PCR analysis was done on equal amounts of total RNAs. A green fluorescent protein encoding transcript present downstream of the overexpressed pri-MIRNA was used to efficiently detect the overexpressed transcript. Threshold cycles are indicated and error bars indicate standard deviation of values obtained based on two independent cDNA syntheses (technical replicates). (b) Detection of mature 21-bp Ath-miR859 and Ath-miR774 by Northern blot analysis in the three independent transgenic lines (shown in (a)) over-expressing Ath-MIR859-774 pri-MIRNA in dcl1.9 and in the DCL1.9/dcl1.9 heterozygous siblings. Total RNAs (10 μg/lane) from each sample were blotted and probed with ³²P-labeled standard DNA complementary to U6 RNA (loading control) or modified oligonucleotides complementary to mature miRNA sequences. (c) Real-time RT-PCR analysis of the relative accumulation of selected Ath-MIR859-774 validated targets (Ath-miR859 target TAIR:At3g49510 and Ath-miR774 target TAIR:At3g19890) in the three transgenic lines (used in (a, b) overexpressing the corresponding pri-MIRNA in dcl1.9 and in the related DCL1.9/dcl1.9 heterozygous background. The histogram represents amounts of specific PCR amplification products (verified by sequencing and dissociation curve analyses) normalized to reference genes [72] defined using Genorm software [73] (see Materials and methods). Error bars indicate the standard deviation of values obtained based on three independent cDNA syntheses (technical replicates).