Processing of Pre-microRNAs by the Dicer-1–Loquacious Complex in Drosophila Cells
Figure 6
Loqs Stimulates the Specific Processing of Pre-miRNA by Dicer-1
(A) Flag–Dicer-1 complex was purified under a harsh condition. Protein extract was prepared from S2 cells that expressed both full-length myc-tagged Loqs (myc-Loqs) and Flag-tagged Dicer-1 (Flag-Dcr-1). The amounts of Loqs in Flag–Dicer-1 complexes prepared under high-salt condition and low-salt condition were examined by Western blotting using anti-myc antibody, which show that less Loqs was co-purified with Dicer-1 in the high-salt condition.
(B) The miRNA processing activities of Flag–Dicer-1 (Flag-Dcr-1) complexes in (A). Flag–Dicer-1 complex containing less Loqs showed a lower activity for the processing.
(C) Recombinant Loqs stimulates the in vitro processing of pre-miR-ban by Flag–Dicer-1 complex purified in high-salt condition. 100 ng of purified GST or GST–Loqs (see Figure 3C) were supplemented for the processing activity by Flag–Dicer-1 complex. GST–Loqs by itself does not show any pre-miRNA processing activity.
(D) GST–Loqs inhibits the siRNA-generating activity of Dicer-1. Uniformly labeled long dsRNA was incubated with Flag–Dicer-1 (Flag-Dcr-1) complex purified in high-salt condition in (A) with or without GST–Loqs. The Flag–Dicer-1 complex by itself showed a considerable activity of generating siRNA from long dsRNA. Addition of GST–Loqs, but not GST, inhibited the processing. Note that the Flag–Dicer-1 (Flag-Dcr-1) complex does not contain Dicer-2 (Dcr-2), judged by Western blot analysis using anti-Dicer-2 antibodies (right panel).