Figure 6.

Effect of pre-treatment with APE1 on Dicer's ability to process 5’ GG-pre-hsa-miR-10b. (A) 350 fmoles of 5’ -³²P-radiolabeled 5’ GG-pre-hsa-miR-10b were treated with purified APE1 (lane 3, left panel gel) for 5 min at 37°C under the in vitro endonuclease assay condition as described in the Materials and Methods. For reference, an alkaline hydrolysis ladder was generated (lane 2, left panel gel; lane 1, right panel gel). For the left panel gel, numbering on the left indicates guanosine residue sites cleaved by RNase T1 under denaturing conditions (lane 1), whereas numbering on the right indicates sites cleaved by APE1. For the right panel gel, the major cleavage products 1 and 2 generated by Dicer are shown with numbering on the right. 5’ -³²P-radiolabeled 5’ GG-pre-hsa-miR-10b pre-treated with (lane 4, right panel gel) or without (lane 3, right panel gel) APE1 prior to incubation with 0.25 units of Dicer for 30 min at 37°C. (B) The RNA secondary structure of 5’ GG-pre-hsa-miR-10b shows the locations of cleavage sites generated by APE1. Dicer products 1 and 2 are shown.