The DGCR8 RNA-Binding Heme Domain Recognizes Primary MicroRNAs by Clamping the Hairpin

Abstract

SUMMARY Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ~30-bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds.

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

“…Although so far little is known on how the selectivity of ADAR1 binding to different pri-miRNAs is achieved, these observations are consistent with the notion that ADAR1 prefers to bind dsRNAs, but with little sequence specificity. Importantly, it was recently reported that two DGCR8 dimers clamp a pri-miRNA hairpin using their RNA-binding heme domain to activate DROSHA cleavage [52] (Figure 6C), thus the ADAR1 binding to pri-miRNA hairpin can compete with DGCR8 to inhibit pri-miRNA processing. Finally, as no A-to-I editing was identified in primary miR302s (data not shown), it further suggests that ADAR1 binding may not always lead to adenosine deamination; for instance, ADAR1 may bind to dsRNAs formed across introns and promote exon circularization [55].…”

“…The iCLIP clusters are depicted as purple rectangles. (C) Two DGCR8 dimers clamp a pri-miRNA hairpin and activate Drosha cleavage (middle) [52]. The binding of ADAR1 to hairpins of pri-miR302s competes with DGCR8 binding, resulting in an inhibitory effect on pri-miR302 processing (right).…”

“…Our iCLIP datasets revealed that both Flag-ADAR1 and Flag-ADAR1-E912A bound directly to multiple apical regions of the hairpin of pri-miR302s ( Figure 6B), consistent with the notion that ADAR1 is a dsRNA-binding protein. As the direct binding of DGCR8 to apical junctions of pri-miRNAs is essential for processing and that two DGCR8 dimers clamp a pri-miRNA hairpin using their RNA-binding heme domain to activate DROSHA cleavage [52,53] (Figure 6C), the binding of ADAR1 to the same stem region of pri-miR302s sug- ) pre-miRNAs were fractionated according to their sizes and were then used for RT-qPCR to quantify the relative abundance of pri-or pre-miR302a. Mature miRNA was measured from total RNAs with miScript II RT-qPCR.…”

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

“…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].…”

Regulation of heme biosynthesis and transport in metazoa

“…Heme inhibits the interaction between P53 and its target DNA element and facilitates the nuclear export and proteasomal degradation of P53 [11]. Heme is also required for the proper binding of the microprocessor subunit DGCR8 to primary microRNA transcripts (pri-miRNAs), and thus plays a critical role in the processing of pri-miRNAs [12][13][14].…”