Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Human polynucleotide phosphorylase (hPNPase), encoded by the PNPT1 gene, is crucial for mitochondrial RNA processing, including import, degradation, and R-loop homeostasis.
• Biallelic mutations in PNPT1 are linked to severe genetic conditions such as hereditary hearing loss and Leigh syndrome.

Purpose of the Study:

• To investigate the functional impact of pathogenic PNPT1 mutations using both bacterial and human cell models.
• To correlate in vitro enzymatic activity with in vivo cellular phenotypes and disease severity.

Main Methods:

• Utilized an Escherichia coli model to assess the effects of four disease-associated PNPT1 mutations.
• Generated a human 293T cell line model using CRISPR-Cas9 gene editing to introduce specific PNPT1 mutations.
• Performed in vitro assays to evaluate RNA binding and degradation activity of wild-type and mutant hPNPase variants.

Main Results:

• Bacterial cells expressing mutant hPNPase alleles displayed phenotypes consistent with loss of function, showing similar responses across different mutations.
• Human cell models exhibited differential responses to distinct PNPT1 mutations, with phenotypes correlating to the clinical severity of the associated pathologies.
• All tested pathogenic mutations impaired protein assembly and affected RNA binding and degradation efficiency to varying extents.

Conclusions:

• The severity of genetic diseases caused by PNPT1 mutations does not directly correlate with the in vitro catalytic activity of the mutant hPNPase proteins.
• Human cellular context is critical for understanding the diverse pathogenic effects of PNPT1 mutations, unlike simplified bacterial models.