Dynamic phosphorylation of DGCR8 regulates embryonic stem cell differentiation and microprocessor function

The Microprocessor complex, minimally composed of DROSHA and DGCR8, is ubiquitously required for microRNA biogenesis. It also regulates the balance between pluripotency and differentiation through mRNA alternative splicing and mRNA cleavage. However, how these various activities are regulated remains incompletely understood. Both DROSHA and DGCR8 are phosphoproteins, and phosphorylation has been reported to influence their molecular functions. In this study, we identified residues in DGCR8 by mass spectrometry that were differentially phosphorylated in mouse embryonic stem cells (mESCs) before and after differentiation into neuronal cells. This included S95 that exhibited greater phosphorylation in undifferentiated cells, and S238 that exhibited greater phosphorylation in differentiated cells. By means of CRISPR/Cas9-mediated genome editing of the endogenous Dgcr8 gene, we generated a homozygous S95A mESC clone and a heterozygous S238A/- mESC clone. Both mutants retained an mESC phenotype but exhibited altered Microprocessor-dependent functions and differentiation outcomes. When directed toward the neuronal lineage, the S238A/- mutant displayed elevated mRNA expression of neuronal progenitor markers even after 10 days, while both S95A and S238A/- mutants failed to generate cardiomyocytes in embryoid body differentiation cultures. Furthermore, MEK1/2 inhibition with a highly selective inhibitor partially phenocopied the transcriptional effects of S95A in mESCs, consistent with a potential contribution of ERK/MAPK signaling for S95 phosphorylation. Together, these findings support a role for site-specific DGCR8 phosphorylation in modulating Microprocessor-dependent processes and differentiation in mESCs, establishing DGCR8 phosphorylation as an important regulatory mechanism in the regulation of Microprocessor activity, especially for stem cell differentiation.