Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing

Abstract

The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity. The reduction causes a dramatic increase in the rate of heme dissociation from DGCR8, rendering the complex labile. Electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies indicate that reduction of the heme iron is accompanied by loss of the cysteines as axial ligands. ApoDGCR8 dimers, generated through reduction and removal of the heme, show low levels of activity in pri-miRNA processing in vitro. Importantly, ferric, but not ferrous, heme restores the activity of apoDGCR8 to the level of the native ferric complex. This study demonstrates binding specificity of DGCR8 for ferric heme, provides direct biochemical evidence for ferric heme serving as an activator for miRNA maturation, and suggests that an intracellular environment increasing the availability of ferric heme may enhance the efficiency of pri-miRNA processing.

Fig. 1.

Reduction of the Fe(III) heme in human NC1 diminishes pri-miRNA processing. (A) Domain structure of human DGCR8. The dsRBDs and C-terminal tail (CTT) are required for cooperative association with pri-miRNAs and for triggering cleavage by Drosha. The human NC1, NC9, and frog DGCR8 HBD-His₆ proteins used in this study are represented by brackets. (B) Electronic absorption spectra of NC1 (7.1 μM) recorded before (dashed line) and after (solid line) incubation with solid dithionite at 25 °C under Ar(g) for 60 min. (C) Electronic absorption spectra of NC1 (10 μM) reduced at pH 6.0 (50 mM MES) using 2 mM dithionite at room temperature under anaerobic conditions for 70 min. (D) Reconstituted pri-miR-30a processing assays were performed at 37 °C for 45 min using recombinant His₆-Drosha^390–1374 and various forms of DGCR8 as indicated. The Fe(III) and Fe(II) NC1 dimers were present at 25 nM and the NC9 monomer concentration was 100 nM. The relationship between the low molecular weight marker (LMWM) and the Decade RNA ladder was inferred from a comparison on a similar gel (Fig. S3).

Fig. 2.

Reduction of the heme iron in DGCR8 greatly decreases the stability of the heme–protein complex. (A) Size exclusion chromatograms of Fe(III) and Fe(II) NC1 at pH 6.0 (50 mM MES). Fe(II) NC1 was prepared as described in Fig. 1C. (B) Electronic absorption spectra of frog HBD (8 μM) at pH 8.0 [50 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS)]. The Fe(III) form of the protein (dotted line) was reduced to the Fe(II) form (solid line) through addition of solid dithionite for 60 min. Subsequently, 66 μL of 80 μM apomyoglobin was added to 200 μL of the Fe(II) HBD solution (dashed line, normalized to compensate for dilution). (C) Fe(III) frog HBD (7 μM) was incubated with a sixfold excess of apomyoglobin at room temperature. The absorbance at 450 nm [Fe(III) DGCR8] and 409 nm (metmyoglobin) are plotted.

Fig. 3.

The Fe(II) heme in frog HBD is a mixture of five-coordinate, high-spin and six-coordinate, low-spin species without a cysteine thiolate ligand. (A) The electronic absorption spectrum (Upper) of Fe(II) frog HBD (11.7 μM) in 50 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) (pH 8.0), 400 mM NaCl, and 1 mM sodium dithionite at 37 °C. MCD spectra (Lower) of the Fe(II) frog HBD (29.4 μM) in 20 mM EPPS (pH 8.0), 160 mM NaCl, 60% (vol/vol) glycerol, and 7 mM sodium dithionite at 4, 8, 15, 25, and 50 K. (Inset) The magnetic saturation behavior of the MCD C-term intensity at 442 nm taken at 2.5, 4.0, 8.0, 15, and 25 K. (B and C) Resonance Raman spectra are shown for Fe(III) (dotted line) and Fe(II) (solid line) frog HBD for low energy (B) and high energy (C) regions. Fe(III) HBD (153 μM) and Fe(II) HBD (136 μM) samples were in 45 mM EPPS (pH 8.0), 360 mM NaCl, and 10% (vol/vol) glycerol; Fe(II) HBD also contained 7 mM sodium dithionite. For the Fe(III) protein, spectra were acquired by excitation with a 457.9-nm line with 20 mW of power at the sample; for the Fe(II) protein, spectra were acquired by excitation with a 413.1-nm line with 14 mW of power at the sample.

Fig. 4.

Fe(III), not Fe(II), heme reconstitutes DGCR8 for pri-miRNA processing in vitro. (A) Electronic absorption spectra of apoNC1 (dotted line), prepared as described in Materials and Methods, and after incubation with equimolar Fe(III) (solid line) or Fe(II) (dashed line) heme. The protein solutions contained 50 mM MES (pH 6.0), 400 mM NaCl, and 1 mM DTT. (B–D) Uniformly ³²P-labeled pri-miR-30a (B), pri-miR-21 (C), and pri-miR-380 (D) were incubated at 37 °C for 45 min, with either Drosha^390–1374 alone (lanes 2, 9, and 16) or Drosha^390–1374 together with various forms of NC1 as indicated (lanes 3–7, 10–14, and 17–20). All reactions were performed in anaerobic conditions, except the ones analyzed in lanes 7 and 14. (E) Filter-binding assays demonstrate that apoNC1 associates with pri-miR-30a with lower cooperativity than that of the Fe(III) heme-bound NC1 (18). The data were best fit using a cooperative dimer model. (F) Hill plot of data from E around the binding transition. The result shown is one of three experiments. K is defined as 10^{(x-intercept)}. Low molecular weight marker, LMWM.