Functional Anatomy of the Human Microprocessor

Abstract

MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…Processing of miRNAs also differs. In animals, the stem-loop structure residing in the pri-miRNA is recognized by the microprocessor, a complex consisting of the RNase III enzyme Drosha and an RNA-binding protein [10–13]. Drosha endonucleolytically cleaves out the stem-loop structure, the precursor-miRNA (pre-miRNA), which is exported out of the nucleus by Exportin-5 [14].…”

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

“…1b), and triggers pri-miRNA cleavage by Drosha. Importantly, the Rhed makes critical contributions to the RNA binding specificity by contacting the stemsingle-strand junctions [11,14], which are known to be important for recognition of a pri-miRNA hairpin [76][77][78]. Removal of heme from NC1 does not appear to alter the affinity for pri-miRNA, but the complexes formed are different from those with Fe(III) heme bound [11].…”

“…Drosha alone has no specific pri-miRNA cleavage activity, because it requires its partner DGCR8 for substrate recognition [6,7]. DGCR8 binds to key structural elements in pri-miRNAs; higher order structures of DGCR8 and Drosha appear to be required for Drosha-mediated cleavage [7][8][9][10][11][12][13][14].…”

Drosha and Dicer: Slicers cut from the same cloth

“…The dsRBDs of DROSHA have weak RNAbinding capacity, which is augmented by DGCR8 (domain architecture in Figure 1A, bottom panel), such that DROSHA and DGCR8 together form a complex termed Microprocessor [1,2]. The binding stoichiometry of Microprocessor has been elucidated from biochemical assays to consist of a heterotrimer composed of one DROSHA and two DGCR8 molecules [3]. The central RNA-binding heme domain (Rhed) of DGCR8 contributes to dimerization and Microprocessor processing fidelity [4], while the dsRBDs of DGCR8 have higher binding affinity to RNA and the C-terminal tail region (CTT) is known to stabilize DROSHA.…”