Crystal structure of dimeric human PNPase reveals why disease-linked mutants suffer from low RNA import and degradation activities

Area of Science:

• Biochemistry
• Molecular Biology
• Mitochondrial Biology

Background:

• Human polynucleotide phosphorylase (PNPase) is a mitochondrial exoribonuclease crucial for RNA turnover and import.
• Impaired PNPase function disrupts mitochondrial RNA import, leading to dysfunction and disease.

Purpose of the Study:

• Investigate the structural and functional consequences of disease-linked PNPase mutations.
• Determine how mutations affect PNPase's RNA binding and degradation activities.
• Elucidate the role of specific PNPase domains in structured RNA binding.

Main Methods:

• Site-directed mutagenesis to create Q387R and E475G PNPase mutants.
• Biochemical assays to measure RNA binding and degradation.
• X-ray crystallography to determine the structure of dimeric PNPase.
• Small-angle X-ray scattering (SAXS) to analyze protein assembly.

Main Results:

• Disease-linked PNPase mutants (Q387R, E475G) form dimers instead of trimers.
• Dimeric PNPase exhibits significantly reduced RNA binding and degradation activity.
• The S1 domain is essential for binding structured RNAs, but these domains are inaccessible in the dimer.
• Crystal structure reveals altered K homology and S1 domain accessibility in the dimeric form.

Conclusions:

• Mutations at the trimeric interface destabilize PNPase into a dimeric form.
• The dimeric structure impairs both RNA import and degradation functions.
• This leads to mitochondrial dysfunction and associated disorders.