Figure 4

Ath-MIR859-774 encodes a functional polycistronic pri-MIRNA. (a) Overexpression of Ath-MIR859-774 pri-MIRNA (under the control of a 35S-CaMV promoter) in wild-type Col-0 transgenic lines (seven representative OverExpressing (OE) lines) is shown. Real-time RT-PCR analysis was performed based on equal amounts of total RNAs. A green fluorescent protein encoding transcript present 3' to the overexpressed pri-MIRNA was used as a tag to efficiently amplify 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 two representative independent transgenic lines (selected from (a)) overexpressing the Ath-MIR859-774 pri-MIRNA. Total RNAs (10 μg/lane) from each sample were blotted and probed with ³²P-labeled standard DNA complementary to U6 RNA (loading control) or 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 two transgenic lines used in (b). 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). Values of target expression in the control line (Col-0) are set to 1. Error bars indicate the standard deviation of values obtained based on three independent cDNA syntheses (technical replicates).