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

Abstract

SUMMARY 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 ribonuclease, 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.

The Genetics and Epigenetics of 22q11.2 Deletion Syndrome

“…In the post-natal period, managing physiological stress should improve vulnerabilities due to miRNA dysregulation, arising from the reduced levels of DGCR8. The identification of small drugs able to modulate the consequences of the haploinsufficiency of both TBX1 and DGCR8 suggest new strategies for reducing the clinical penetrance and severity of 22q11.2del (Barr et al, 2015;Fulcoli et al, 2016). In summary, DNA and RNA sequencing approaches are unveiling new genetic and epigenetic modifiers among the 22q11.2del cohort that have a significant impact on disease penetrance and severity.…”

“…The fact that the expression of many distinct miRNAs fluctuate over time and are variable from patient to patient likely explains some of the post-natal complications that differ among 22q11.2del patients (De La Morena et al, 2013). Future directions for such patients may be clinical interventions to improve DGCR8 functions, with recent experiments reporting that protoporphyrins can enhance miRNA biogenesis in DGCR8-haploinsufficient cells (Barr et al, 2015;Sachar et al, 2016).…”

Consequences of 22q11.2 Microdeletion on the Genome, Individual and Population Levels

“…[ 41 , 42 ]. Recent research reports that protoporphyrins can increase miRNA biogenesis in DGCR8 haploinsufficient in in vitro mouse cells, so this could be potentially therapeutic for 22q11.2DS patients [ 43 ]. Other fragments embedded in the deleted segment that could affect the clinical presentations are long noncoding RNAs (lncRNA) and small nuclear RNAs (snoRNAs).…”

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

“…The functional importance of heme in a native coordination environment was probed by replacement of heme with Co(III) protoporphyrin IX (PPIX) in cellulo in heme deficient HeLa cells and in vitro with Co(III)PPIX-loaded NC1. Microprocessor activity is nearly the same in Co(III)-loaded protein as in the natively heme-loaded protein [79]. Co(III)PPIX-reconstituted NC1 exhibits a hyperporphyrin spectrum, suggesting that there are two bound Cys(thiolate) ligands in the Co(III) NC1 protein.…”