Figure 2.

Ribonuclease structure mapping is consistent with the most probable secondary structure resulting from the SHAPE-constrained MC-Fold calculations for (A) pri-mir-16-1, (B) pri-mir-30a, and (C) pri-mir-107. For each RNA, a denaturing 12% polyacrylamide gel used in the analysis is shown, with lanes as follows: C is a control sample (no nuclease); OH⁻ is a limited alkaline digest; and T1, A, and V1 are limited digests with ribonucleases specific for single-stranded G, single-stranded C and U, and 5′ to double-stranded or well stacked single-stranded regions, respectively. The reactions in lanes 2 and 3 were performed under RNA-denaturing conditions (denoted ‘Den.’) in order to provide a ladder correlating position in the gel with the nucleotide sequence; while the reactions in lanes 4–6 were performed under RNA-native conditions (denoted ‘Nat.’). Helical and loop regions of the RNA are indicated to the right of the gel. The highest probability secondary structure (see Fig. 1) with positions of cleavage by ribonucleases under native conditions indicated by symbols as described in the legend is displayed below each gel. Symbol size is proportional to cleavage intensity. In these secondary structure maps, proposed Drosha cleavage sites are identified with red arrows; regions near Drosha cleavage sites displaying high single-strand probability in our MC-Fold and SHAPE analysis are enclosed in grey boxes.