Figure 6

Schematic representation of the MIR156g gene and its precursors. Detection of pri-, pre- and mature miR156g. (A) MIR156g gene structure; thin black vertical bars within exons show additional splice sites identified during pri-miRNA156g analyses; dotted-vertical lines within the last exon together with pA symbols denote polyadenylation sites. (B) pre-miRNA156g hairpin structure (ΔG=−65.85 kcal/mol) and its rice orthologue (ΔG=−61.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1. (C) Structures of splice isoforms (I–VIII) of the miR156g transcript; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. (D) RT-PCR analysis of first intron retention throughout barley plant life stages. (E–F) pri-miRNA156g RT-PCR expression analysis in five barley developmental stages. Arrows on agarose gel indicate splice isoforms II, III and V. (G) Real-time PCR measurements of total pri-miRNA156g expression levels (upper graph) and pri-miR156g fragments carrying the first intron (+IVS1) and after the first intron splicing (ΔIVS1) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. (H) Nucleotide sequence of the mature miR156g molecule, and detection of pre-miRNA and mature miR156g using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1. Colors, abbreviations, and symbols as in Figure 1. Additional colors depict alternatively spliced exons in the pri-miRNA.