Construction and characterization of ribonuclease H2 C subunit-knockout NIH3T3 cells

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mammalian ribonuclease (RNase) H2 is a heterotrimeric enzyme essential for DNA repair.
• RNase H2 removes ribonucleotides misincorporated into genomic DNA.
• Mutations in RNase H2 genes are linked to Aicardi-Goutières syndrome (AGS), a neuroinflammatory disorder.

Purpose of the Study:

• To investigate the role of the RNase H2 C subunit (RH2C) in ribonucleotide removal from genomic DNA.
• To characterize the functional impact of AGS-associated RH2C mutations.
• To compare the effects of RH2C deficiency with previous findings on RNase H2 A subunit (RH2A) deficiency.

Main Methods:

• Construction and characterization of RH2C-knockout mouse fibroblast NIH3T3 cells.
• Assay of ribonucleotide-hydrolyzing activity in genomic DNA.
• Analysis of ribonucleotide accumulation in genomic DNA.
• Transient expression of wild-type and mutant RH2C in knockout cells.

Main Results:

• RH2C-knockout cells showed significantly reduced ribonucleotide-hydrolyzing activity.
• Genomic DNA from knockout cells accumulated higher levels of ribonucleotides.
• Expression of wild-type RH2C restored normal activity and reduced ribonucleotide accumulation.
• Expression of AGS-associated RH2C variants (R69W, K145I) failed to fully restore function, mirroring effects seen with RH2A mutations.

Conclusions:

• The RNase H2 C subunit is critical for maintaining genomic integrity by removing ribonucleotides.
• AGS-causing mutations in RH2C impair its catalytic function, contributing to disease pathogenesis.
• These findings highlight the conserved importance of both RNase H2 A and C subunits in preventing DNA damage and associated neuroinflammation.