FIGURE 4.

Visualization of miRLC and miRISC on native agarose gels. (A) pre-let-7 was labeled with ³²P and incubated in S2 cell lysates in the presence of EDTA for 3 min and 30 min. After 30 min of incubation, Mg²⁺ was added to the reaction mixture to induce DCR-1 activity. Further incubation was carried out as indicated. The resultant samples at each time point were loaded onto an agarose gel, and complexes containing either pre-let-7 or mature let-7 were visualized after running the gel. Simultaneously, siRNA duplex labeled with ³²P was incubated in the S2 lysates in the presence of EDTA or Mg²⁺. After 30 min of incubation, both the lysates were loaded on the same gel system. The input lanes on the far left and right sides show pre-miRNA and siRNA duplexes by themselves, respectively. The bands with an asterisk and two asterisks correspond to miRLC and miRISC, respectively. The bands corresponding to siRLC and siRISC are as indicated on the gel. The band shown with a black triangle is an unknown product, which did not appear in previous equivalent experiments (Miyoshi et al. 2005) but appeared in this study. (B) The bands indicated as miRLC and miRISC in A were isolated from the gel, and RNAs isolated were subjected to a native acrylamide gel. The miRLC contained pre-let-7, whereas miRISC contained mostly single-stranded let-7. (Input lane) Contains pre-let-7 as the starting material. Pre-let-7 incubated in S2 lysates was also subjected to the same gel (S2 lysate + pre-let-7), which shows the migration patterns of pre-let-7, let-7/let-7*, and mature let-7.