Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene

Abstract

Maturation of microRNAs (miRNAs) begins by the “Microprocessor” complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other “noncanonical” functions of the DGCR8 protein.

DGCR8haploinsufficiency leads to primate-specific RNA dysregulation and pluripotency defects

“…We observed that a third of the miRNAs that are affected by DGCR8 haploinsufficiency are primate-specific and not present in rodents, some of which have been shown to be associated with pluripotency maintenance and self-renewal, including the C19MC cluster (Lin et al , 2010; Mong et al ., 2020). Ree et al has also reported defects in C19MC miRNA processing, despite an inconsistent efficiency in targeting DGCR8 expression in human cells (Reé et al , 2022). The clonal expansion defects observed in DGCR8 HET hESCs could be rescued by reintroducing four independent miRNAs belonging to this cluster, indicating its contribution to proliferation.…”