Post-transcriptional control of DGCR8 expression by the Microprocessor - PubMed (original) (raw)
Post-transcriptional control of DGCR8 expression by the Microprocessor
Robinson Triboulet et al. RNA. 2009 Jun.
Abstract
The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.
Figures
FIGURE 1.
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.
FIGURE 2.
DGCR8 expression is regulated by the Microprocessor. Hela cells were transfected with either scrambled (Sc), DGCR8, or Drosha siRNA as indicated. Cells were collected for analysis 60 h post-transfection. (A) Whole cell extracts were analyzed by Western blot using anti-Drosha, DGCR8, and Tubulin antibodies. (B) Total RNA was analyzed by quantitative RT-PCR using primers specific for Drosha, DGCR8, and Actin mRNA. Error bars represent SEM with N = 3. (C) Schematic representation of the firefly luciferase reporter containing DGCR8 5′UTR (pGL3c DGCR8 5′UTR). (D) Hela cells were co-transfected with either pGL3c or pGL3c DGCR8 5′UTR plasmid together with a TK-Renilla luciferase plasmid. Luciferase activity was measured 48 h post-transfection. Firefly luciferase activity was normalized relative to that of Renilla luciferase. Error bars represent SEM with N = 3. (E) Hela cells were transfected with pGL3c DGCR8 5′UTR together with TK-Renilla luciferase vector, and either scrambled, DGCR8, or Drosha siRNA as indicated. Luciferase activity was measured 60 h post-transfection. Renilla luciferase activity was used for normalization. Error bars represent SEM with N = 5.
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