Figure 2

Analysis of ast-mir-76, a miRNA that was previously unknown in mosquitoes. The mature miRNA sequence was cloned from An. stephensi, and the hairpin precursor sequence was obtained from the An. gambiae genome assembly. A) Alignment of the pre-miRNA hairpins found in An. gambiae and Ae. aegypti. The mature miRNA is marked in red while miRNA* is marked in blue. Conserved nucleotides are indicated by a "+". B) Hairpin structure of An. gambiae mir-76. The mature miRNA predicted by miRscan (Table 2) is shown in red. C) RPA analysis of ast-mir-76. Lane 1, An. stephensi RNA with probe and digested; Lane 2, yeast RNA with probe and digested; Lane 3, yeast RNA without probe and digested; Lanes 4 and 5, empty lanes; Lane 6, undigested probe. A band of the correct size was only observed in An. stephensi total RNA samples (Lane 1). The size of the protected RNA product in lane 1 was estimated to be 24 nucleotides using markers as described in Figure 1. This size is as expected (the protected 20-nt long ast-miR-76 plus 4 undigested adenosines, see Methods).