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Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene
Dóra Reé1,2, Ábel Fóthi1, Nóra Varga1
1Institute of Enzymology, Research Center for Natural Sciences, 1117 Budapest, Hungary.
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.
Insights
DiGeorge Syndrome Critical Region 8 (DGCR8) heterozygous mutation in human embryonic stem cells reduces DGCR8 protein levels but not self-renewal or differentiation. This model aids study of DGCR8
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- MicroRNA (miRNA) maturation is initiated by the Microprocessor complex, comprising Drosha and DiGeorge Syndrome Critical Region 8 (DGCR8).
- DGCR8 has known roles in miRNA processing and emerging evidence suggests miRNA-independent functions in RNA regulation and tissue development.
Purpose of the Study:
- To investigate the specific cellular and molecular roles of DGCR8.
- To characterize a human embryonic stem cell (hESC) line with a monoallelic DGCR8 mutation for studying DGCR8 functions.
Main Methods:
- CRISPR-Cas9 gene editing to create a monoallelic DGCR8 mutation in hESCs.
- Genetic characterization of single-cell derived clones.
- Assessment of DGCR8 mRNA and protein levels.
- Evaluation of hESC self-renewal and trilineage differentiation capacity.
- Analysis of pri-miRNA processing.
Main Results:
- DGCR8 heterozygous mutation led to modest mRNA level changes but significant protein reduction.
- hESC self-renewal and trilineage differentiation remained unaffected.
- Partial disruption of Microprocessor function in pri-miRNA processing was observed in some clones.
Conclusions:
- The developed DGCR8 mutant hESC line is a valuable model for studying both canonical miRNA biogenesis and noncanonical DGCR8 functions.
- DGCR8 protein levels are critical for its functions, even with preserved mRNA levels.
- The study highlights the complexity of DGCR8 roles beyond primary miRNA processing.
