Cobalt(III) Protoporphyrin Activates the DGCR8 Protein and Can Compensate microRNA Processing Deficiency

Abstract

Processing of microRNA primary transcripts (pri-miRNAs) is highly regulated and defects in the processing machinery play a key role in many human diseases. In 22q11.2 deletion syndrome (22q11.2DS), heterozygous deletion of DiGeorge critical region gene 8 (DGCR8) causes a processing deficiency, which contributes to abnormal brain development. The DGCR8 protein is the RNA-binding partner of Drosha RNase, both essential for processing canonical pri-miRNAs. To identify an agent that can compensate reduced DGCR8 expression, we screened for metalloporphyrins that can mimic the natural DGCR8 heme cofactor. We found that Co(III) protoporphyrin IX (PPIX) stably binds DGCR8 and activates it for pri-miRNA processing in vitro and in HeLa cells. Importantly, treating cultured Dgcr8(+/-) mouse neurons with Co(III)PPIX can compensate the pri-miRNA processing defects. Co(III)PPIX is effective at concentrations as low as 0.2 μM and is not degraded by heme degradation enzymes, making it useful as a research tool and a potential therapeutic.

Figure 1

Metalloporphyrins that were tested for association with DGCR8 and for activation of pri-miRNA processing. (A) Schematic of a metalloprotoporphyrin IX. The M at the center represents a metal ion. (B) A section of the periodic table showing the elements used in this study, with the group numbers labeled. The protoporphyrin IX complexes with the metals shown in red background stably bind to DGCR8, whereas the complexes with the elements in gray do not.

Figure 2

Protoporphyrin IX without a metal center does not stably associate with apoNC1. (A) Electronic absorption spectra of titration of PPIX into 1.25 μM apoNC1 dimer at 0.25 μM intervals, in 50 mM MES pH 6.0, 400 mM NaCl and 1 mM DTT The PPIX:apoNC1 ratios of the bold curves are indicated in italic. (B) Absorbance values at 368 nm and 447 nm obtained from the titration are plotted against PPIX:apoNC1 ratios. To avoid overlapping, the A₃₆₈ values are nudged up by 0.01. In contrast to a similar titration experiment using Fe(III)PPIX (Barr, et al., 2011), no saturation point is observed at 1:1 PPIX:apoNC1 ratio, indicating a lack of specific binding. (C) Size exclusion chromatogram of a mixture of apoNC1 and PPIX, both at 4 μM concentration shows little co-elution. The low 368 nm absorption in the elution peak was contributed by the ~10% heme remaining bound to the apoNC1 preparation. The chromatogram is indistinguishable from that of the apoNC1 alone. See also Figure S1.

Figure 3

Co(III)PPIX, but not the Co(II) form, associates with apoNC1. (A) Electronic absorption spectra of Co(III)PPIX titrated into 2 μM apoNC1 dimer at 1 μM per step, in 50 mM MES pH 6.0, 400 mM NaCl and 1 mM DTT. The absorption peak at 432 nm results from a non-specific interaction. An absorbance spectrum of Co(III)PPK alone is shown as a dashed line. (B) Size exclusion chromatogram of the reconstituted Co(III)PPIX-NC1 complex. (C) Co(II)PPIX was titrated into 2.5 μM apoNC1 in 50 mM MES pH 6.0, 400 niM NaCl and 2 mM sodium dithionite at 1.25 μM per step. Sodium dithionite obscures peaks below 350 nm due to its high absorbance. The apoNC1 spectrum before addition of sodium dithionite is also shown. The absorption spectrum of Co(II)PPIX without proteins is shown in the inset. (D) Size exclusion chromatogram of apoNC1 with Co(II)PPIX. The SEC buffer contained 50 mM MES pH 6.0 and 400 mM NaCl, and was degassed to remove O₂. The apoNC1 dimer elutes at an expected volume. However, there is no large accompanying absorbance at 398 nm (the Co(II)PPIX Söret wavelength) or 456 nm (potential re-oxidized Co(III)PPIX Söret wavelength). See also Figures S2 and S3 and Table S1.

Figure 4

Co(III)PPIX activates DGCR8 for pri-miRNA processing in vitro. Denaturing gel analyses of cleavage assays of (A) pri-miR-23a, (B) pri-miR-21, (C) pri-miR-380 and (D) pri-miR-30a. The cleavage reactions contained trace amounts of uniformly ³²P-labeled pri-miRNAs, recombinant His6-Drosha^390–1374 and the various forms of DGCR8 proteins (25 nM dimer). NC1 is the native Fe(III) heme-bound dimer. apoNC1 is NC1 with the >90% of heme removed. The reactions labeled with “+Fe(III)PPIX” and “+Co(III)PPIX” contained apoNC1 incubated with equimolar of Fe(III) heme or Co(III)PPIX, respectively. Fractions of pri-miRNAs converted to pre-miRNAs are plotted as means ± SD (n = 3 for pri-miR-23a, pri-miR-21 and pri-miR-380; n = 4 for pri-miR-30a). Asterisks indicate statistically significant activation of apoNC1 (**, p ≤ 0.01; *, 0.01< p ≤ 0.05).

Figure 5

Live-cell pri-miRNA processing assay shows that Co(III)PPIX activates pri-miRNA processing without inducing cytotoxicity. Hela cells were cultured in heme-depleted media, transfected with the pri-miR-9-1 reporter, treated for 10 h with succinylacetone (1 mM) either alone or together with Co(III)PPIX or hemin at the indicated concentrations. (A) Normalized eYFP/mCherry fluorescence slopes (± 95% CI). (B) Abundance of mature miR-9 normalized by that of β-actin mRNA (mean ± SD, n = 4). (C) MTT assays showed little cytotoxicity (mean ± SD, n = 4). (D–F) Reporter assays were performed similarly to described above, except that the N-flag-DGCR8 expression plasmid was cotransfected with the reporters, that the pri-miR-9-1 (D), pri-miR-185 (E), and pri-miR-30a (F) reporters were used, and that SA, Co(III)PPIX and hemin were added prior to transfection. See also Figure S4.

Figure 6

Co(III)PPIX restores deficient miRNA expression caused by heterozygous deletion of the Dgcr8 gene in mice. Primary cortical neurons dissected from Dgcr8^+/− mouse embryos and their wild-type littermates were treated with Co(III)PPIX at the indicated concentrations between DIV 1 and 3, for 48 h. (A,B) Mature miR-185 (A) and miR-134 (B) levels after normalization to that of the Gapdh mRNA. The miR-134 data at lower Co(III)PPIX concentrations did not reach statistical significance and thus are not shown. Each plotted value is average ± SEM from seven independent experiments using seven animals for each genotype. Select P values are calculated using two-tailed Student’s t-test and are indicated on the graph. (C) Images of the treated and control neurons with processes visualized via MAP2 staining (shown in green).