Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing

Abstract

DGCR8/Pasha is an essential cofactor for Drosha, a nuclear RNase III that cleaves the local hairpin structures embedded in long primary microRNA transcripts (pri-miRNAs) in eukaryotes. Although our knowledge of pri-miRNA processing has significantly advanced in recent years, the precise role of DGCR8 in this pathway remains unclear. In our present study, we dissect the domains in DGCR8 that contribute to the processing of pri-miRNAs and the subcellular localization of DGCR8. Drosha is stabilized through an interaction between its middle domain and the conserved C-terminal domain of DGCR8. Furthermore, DGCR8, but not Drosha, can directly and stably interact with pri-miRNAs, and the tandem dsRNA-binding domains (dsRBDs) in DGCR8 are responsible for this recognition. Moreover, the DGCR8 N-terminal region upstream of its dsRBDs is unnecessary for pri-miRNA processing but is critical for nuclear localization. Our study thus provides further insights into the mechanism of action of the Drosha–DGCR8 complex in pri-miRNA processing.

Processing of microRNA primary transcripts requires heme in mammalian cells

“…1D). The C-terminal tail of DGCR8 has been shown to be required for pri-miRNA processing (31,32), and a mutant with the C-terminal tail deleted (ΔCTT) was used as an inactive control (Fig. 1D).…”

Structure of the dimerization domain of DiGeorge Critical Region 8

“…Previous studies showed that (a) the dsRBDs and CTT regions of DGCR8 (residues 499-751) are monomeric in the absence of RNAs and are sufficient for pri-miRNA processing 18,24 ; (b) the DGCR8 406-751 is also monomeric but is inactive, and therefore, the immediate N-terminal neighboring region (residues 406-498) of dsRBD1 is an autoinhibition domain (Gong et al manuscript provided with this submission); and (c) NC1 (residues 276-751), containing the dimerization and autoinhibition domains (they form the HBD), dsRBDs and CTT, can form heme-bound dimer that is active in primiRNA processing. 18 Thus, the dimerization domain we characterized in this study can activate miRNA maturation through releasing the autoinhibition.…”

“…23 The C-terminal portion of the 773-residue DGCR8 protein (residues 499-751) contains two double-stranded RNA-binding domains (dsRBDs) and a conserved C-terminal tail (CTT) that are sufficient for pri-miRNA processing. 18,24 The N-terminal portion of DGCR8 (residues 1-275) is required for nuclear localization. 24 The only recognizable motif in the central region of DGCR8 is a WW motif (residues 307-329), which is a widely distributed protein motif characterized by two tryptophan residues.…”

“…18,24 The N-terminal portion of DGCR8 (residues 1-275) is required for nuclear localization. 24 The only recognizable motif in the central region of DGCR8 is a WW motif (residues 307-329), which is a widely distributed protein motif characterized by two tryptophan residues. 25 WW motifs are often used for interacting with proline-containing sequences.…”

Robustness of the hypoxic response: Another job for miRNAs?

“…They are generally synthesized by the RNA polymerase II, and then processed sequentially by two complexes containing Drosha and Dicer, to be finally incorporated into the RNA-induced silencing complex (RISC). The mature miRNA leads the RISC complex to recognize the target messenger RNA (mRNA) by sequence complementarity, usually found in the 3 0 -untranslated region (3 0 UTR) (Kim, 2005;Yeom et al, 2006;Jaubert et al, 2007;Inui et al, 2010). Generally, miRNAs promote degradation and/or inhibit translation of their target mRNAs (Djuranovic et al, 2011).…”