Heme enables proper positioning of Drosha and DGCR8 on primary microRNAs

Alexander C Partin,Byung-Cheon Jeong,Tri D Ngo,Yunsun Nam,Gary Hon,Emily Herrell

doi:10.1038/s41467-017-01713-y

Alexander C Partin, Byung-Cheon Jeong + Show 4 more

Open Access

https://doi.org/10.1038/s41467-017-01713-y

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Abstract

MicroRNAs regulate the expression of many proteins and require specific maturation steps. Primary microRNA transcripts (pri-miRs) are cleaved by Microprocessor, a complex containing the RNase Drosha and its partner protein, DGCR8. Although DGCR8 is known to bind heme, the molecular role of heme in pri-miR processing is unknown. Here we show that heme is critical for Microprocessor to process pri-miRs with high fidelity. Furthermore, the degree of inherent heme dependence varies for different pri-miRs. Heme-dependent pri-miRs fail to properly recruit Drosha, but heme-bound DGCR8 can correct erroneous binding events. Rather than changing the oligomerization state, heme induces a conformational change in DGCR8. Finally, we demonstrate that heme activates DGCR8 to recognize pri-miRs by specifically binding the terminal loop near the 3′ single-stranded segment.

Highlights

MicroRNAs regulate the expression of many proteins and require specific maturation steps
In order to investigate the role of heme in miR biogenesis, we purified recombinant human Drosha and DGCR8 polypeptides from insect cells and isolated heme-saturated (MPHeme) and hemedeficient (MPApo) species (Fig. 1a and Supplementary Fig. 1a)
Though reminiscent of “abortive processing” described previously for mutant substrates or domain deletions[17,20], what is striking is that wild-type Drosha and DGCR8 reverse their orientation on a wild-type pri-miR, depending on the mere presence of the heme molecule

Summary

Introduction

MicroRNAs regulate the expression of many proteins and require specific maturation steps. Primary microRNA transcripts (pri-miRs) are cleaved by Microprocessor, a complex containing the RNase Drosha and its partner protein, DGCR8. DGCR8 is known to bind heme, the molecular role of heme in pri-miR processing is unknown. We demonstrate that heme activates DGCR8 to recognize pri-miRs by binding the terminal loop near the 3′ single-stranded segment. Assigning Drosha to the basal junction led to a model where DGCR8 recognizes a “UGU” motif in the terminal loop[20,22]. How dimerization, heme binding, and pri-miR processing are mechanistically related is unknown. We show that heme is necessary for MP to cleave certain substrates at the correct site, and the degree of heme dependence varies for different pri-miRs. Heme dependence arises from failure of the basal junction to recruit Drosha; heme-bound a Drosha

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