Table 7_Dynamic phosphorylation of DGCR8 regulates embryonic stem cell differentiation and microprocessor function.xlsx

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.</p>