miRNA-like long hairpins producing phased 21-nt small RNAs. (A) Hairpin of the pre-miR436. In total, 13,141 and 2439 sequence reads were generated from the 5′ and the 3′ arms of the hairpin, respectively, and only 19 reads were mapped to the antisense strand. Of these sequences, 91.2% were 21 nt in length and 95.9% of these 21-nt small RNAs fell in phases of 21-nt spacing. The number of reads for all small RNAs with a common 5′ terminus (regardless of size) is plotted, with red and blue bars representing sequences from the 5′ and the 3′ arms, respectively. The 21-nt phases corresponding to the 5′ arm are indicated by the vertical dotted lines. Phase identifiers are shown above and below the hairpin. Nucleotide positions shown on the X-axis are based on the 5′ arm of the hairpin starting from the 5′ end of phase P12_5′. miR436 and miR436* are shown in green and orange, respectively. (B) A miRNA-like long hairpin generated from the antisense strand of LOC_Os06g21900. In total, 59,150 sequence reads were generated (∼60% from the 5′ arm and ∼40% from the 3′ arm; ∼97% of the reads were from 6–10 DAF grains), including 97 reads mapped to the antisense strand; 54,857 (92.7%) reads were 21 nt in length and 93.9% of these 21-nt small RNAs were in exact 21-nt phases. The frequency and distribution of small RNAs is plotted as described in A. (C) 5′ RACE validation of cleavage of predicted target mRNAs by P6_5′ and P6_3′. (D) Expression of small RNAs from the newly identified long hairpin in different tissues detected by a probe antisense to the 3′ arm of the hairpin (218 nt in length). The blot used was the same as that for miR1861 shown in Figure 1C. (S) Shoots; (R) roots; (G1) 1–5 DAF grains; (G6) 6–10 DAF grains.
