p53 in cytoplasm exerts 3'→5' exonuclease activity with dsRNA

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Double-stranded RNA (dsRNA) is crucial for cellular processes, and its levels are tightly regulated.
• The tumor suppressor protein p53 has known 3'→5' exonuclease activity, but its role in dsRNA metabolism was unclear.

Purpose of the Study:

• To investigate the degradation of dsRNA by the exonuclease activity of the p53 protein.
• To elucidate the connection between p53 and dsRNase activity within the cell cytoplasm.

Main Methods:

• Utilized recombinant purified wild-type p53 (wtp53) and endogenous p53 in cellular fractions.
• Assessed dsRNA degradation by measuring nucleotide removal from dsRNA 3'-ends.
• Employed cell lines with differing p53 statuses (HCT116 p53+/+ and HCT116 p53-/-) and performed transfections with wtp53 and mutant p53.
• Indicated dsRNA-protein complex formation and disruption using anti-p53 antibodies.

Main Results:

• Recombinant and endogenous p53 demonstrated the ability to degrade dsRNA by removing nucleotides from the 3'-end.
• Cytoplasmic dsRNase activity correlated directly with the presence and levels of endogenous p53.
• HCT116 cells expressing p53 showed significantly higher dsRNase activity than p53-deficient cells.
• Transfection with wtp53, but not a nuclease-deficient mutant, restored dsRNase activity in p53-null cells.
• γ-irradiation-induced p53 accumulation in the cytoplasm was accompanied by increased dsRNA degradation.
• dsRNA was shown to form a complex with p53.

Conclusions:

• The p53 protein possesses cytoplasmic dsRNase activity, capable of degrading dsRNA.
• This p53-dependent dsRNA degradation can lead to either complete elimination or the generation of shorter dsRNA fragments.
• The dsRNase function of p53 in the cytoplasm may play roles in regulating translation and inducing apoptosis.