DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

Abstract

DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.

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).…”

The Crosstalk between Micro RNA and Iron Homeostasis

“…This potential role for iron to participate in miRNA biogenesis was first demonstrated when DGCR8 was identified as a heme-binding protein [59]. In reconstituted pri-miRNA processing assays, heme binds to and enhances DGCR8 dimerization, which is required for increased activity of this essential miRNA processing cofactor [60][61][62][63]. During this process, heme binds to DGCR8 via its Fe (III) iron co-factor.…”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…Finally, larger assemblies could be generated by reciprocal binding of distinct pri-miRNAs to individual dsRBDs of the microprocessor. Electron tomographic studies of pri-miRNAs in complex with DGCR8 have produced images of ~400 kDa pri-miRNA:DGCR8 complexes that suggest such an arrangement [21]. Given that many miRNA precursors are transcribed in clusters, this last model may in fact be a biological means to improve the efficiency of pre-miRNA formation.…”