Processing of amiRchs1 and miR319 precursors.

(A) Small RNA sequences from the miRBase database (v20 http://miRbase.org/index.shtml) were incorporated into the predicted stem-loop structure of the Arabidopsis miR319a precursor. Small RNAs cloned fewer than 5 times are also indicated. (B) Small RNA sequences from amiRchs1 transgenic petals incorporated into the scheme for the amiRchs1 precursor. Only small RNAs cloned more than 5 times are indicated. (C) Processing intermediates detected using 5′ RLM-RACE PCR amplification. The positions of cleavage sites, as revealed by 5′ RACE, and the number of sequenced clones corresponding to each site, are indicated by black arrows. The four sites marking the origins of B1 and B2 small RNAs, and corresponding to the four marked fragments in the polyacrylamide gel, are indicated by blue arrows (Sites 1, 2, 3 and 4). Site II corresponds to the cleavage site that marks the origin of the most frequent B1 small RNAs, but the processing intermediates had not been sampled by random sequencing of RACE PCR products. Left inset: Polyacrylamide gel showing fragments after 5′ RACE. (D) Small RNA sequences from amiRchs1 transgenic petals incorporated into the scheme for the petunia miR319a precursor. Only small RNAs cloned more than 5 times are indicated. B1, B1*, B2, and B2* correspond to the regions producing the sequences previously designated as miR319a.1, miR319a.1*, miR319a.2 and miR319a.2* [48], [49], respectively. B3 corresponds to the region between B1 and B2, B3* corresponds to the region between B1* to B2*, and B3+ corresponds to a region longer than B3, in which the 3′ ends of B3 sequences stretched into the middle of B1.