Hairpins in the DGCR8 mRNA are cleaved by the Microprocessor. (A) In vitro processing assays performed with pri-miR-1, DGCR8 5′UTR RNA, or RNA encompassing the miR-1306 hairpin. The indicated in vitro transcribed, internally labeled RNA was incubated with or without Flag-immunopurified DGCR8 (Flag-DGCR8 IP) or Drosha (Flag-Drosha IP). Arrows indicate major processing products. (B) Reconstitution of DGCR8 5′UTR RNA processing activity with the recombinant Microprocessor. RNA was incubated with recombinant DGCR8 (rDGCR8) together with an increasing amount of recombinant Drosha (rDrosha). Flag-Drosha IP served as a positive control. Arrow indicates DGCR8 5′UTR “pre-miRNA-like” processing product. (C) In vitro processing of pre-miR-1 and DGCR8 5′UTR pre-miRNA-like hairpin by Dicer. Gel purified pre-miR-1 and DGCR8 5′UTR hairpin from the in vitro processing assays shown in B were incubated with recombinant Dicer (rDicer). Arrows indicate ∼22 nt duplex and arrowheads indicate the terminal loops processed from the hairpin RNA substrates. RNA was resolved on 15% polyacrylamide denaturing gels and visualized by autoradiography (A–C). (D) Detection of the hairpin processed from the DGCR8 5′UTR in vivo. Hela cells were transfected with either the luciferase reporter plasmid containing the DGCR8 5′UTR depicted in Figure 2C (pGL3c DGCR8 5′UTR) or luciferase reporter plasmid without DGCR8 5′UTR (pGL3c). Total RNA was extracted 48 h post-transfection and analyzed by Northern blot using a probe to specifically detect the DGCR8 5′UTR hairpin.
