Exonucleolytic degradation of RNA by p53 protein in cytoplasm

Abstract

p53 in cytoplasm displays an intrinsic 3'-->5' exonuclease activity. To understand the significance of p53 exonuclease activity in cytoplasm, cytoplasmic extracts of various cell lines were examined for exonuclease activity with different single-stranded RNA (ssRNA) substrates. Using an in vitro RNA degradation assay, we observed in cytoplasmic extracts of LCC2 cells, expressing high levels of endogenous wtp53, an efficient 3'-->5' exonuclease activity with RNA substrates, removing the 3'-terminal nucleotides. Interestingly, RNA containing AU-rich sequences (ARE) is the permissive substrate for exonucleolytic degradation. Evidence that exonuclease function with RNA detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: (1) this activity closely parallels with status and levels of endogenous cytoplasmic p53; (2) the endogenous exonuclease exerts identical RNA substrate specificity and excision profile characteristic for purified baculovirus-or bacterially-expressed wtp53s; (3) the exonuclease activity with ARE RNA is competed out by the presence of ss or double-stranded DNA substrate utilized by p53 protein in cytoplasm; (4) immunoprecipitation by specific anti-p53 antibodies markedly reduced the exonuclease activity with both RNA and DNA substrates; and (5) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into p53-null H1299 or HCT116 cells induced high levels of exonuclease activity with ARE RNA substrate in cytoplasm with characteristic excision profile. The efficient ARE RNA degradation correlates with the efficient binding of p53 to ARE RNA in cytoplasm. The possible role of p53 exonuclease activity in ARE-mRNA destabilization in cytoplasm, which may be important for expression of proteins that control cell growth and/or apoptosis is discussed.

p53 in mitochondria enhances the accuracy of DNA synthesis

“…The contribution of endogenous p53 to the high level of exonuclease activity in Our studies illustrated that cytoplasmic fractions of H1299 or HCT116 cells overexpressing wtp53, but not exonucleasedeficient mutant p53-R175H, had significantly higher levels of exonuclease activity, than those transfected with the control vector, supporting the likelihood that p53 in cytoplasm is linked to observed exonuclease activity. 32 These cytoplasmic fractions were used to further define the contribution of endogenous p53 exonuclease to the error correction in H1299mit. The results illustrate the efficient excision of the incorporated T:G mispair in the presence of H1299cyt overexpressing wtp53 ( Figure 6, lanes 3 and 4).…”

Excision of nucleoside analogs in mitochondria by p53 protein

“…Our studies [35] illustrated that cytoplasmic fractions of H1299 cells overexpressing wtp53, but not exonucleasedeficient mutant p53-R175H, had higher levels of exonuclease activity than those transfected with the control vector. Furthermore, increased removal of incorporated wrong nucleotide was observed in mitochondrial fraction in the presence of H1299cyt overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H [31].…”

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

“…Our previous studies showed that p53 in cytoplasmic extracts displays a high level of 3'→5' exonuclease activity with various DNA and RNA substrates. 11,13 We attempted to analyze the exonuclease activity with dsRNA substrates in the cytoplasmic fractions of cells. The experimental model system that we utilized included non-stressed human breast cancer cells LCC2 (carrying wtp53) and p53-null H1299 cells.…”

“…12 Furthermore, p53 efficiently excises 3'-terminal nucleotide from ssRNA substrates including RNA containing AU-rich sequences, which may be important for the expression of proteins that control cell growth and/or apoptosis. 13 p53 activation caused by DNA-damaging stress results in a substantial increase in the expression of protein kinase R (PKR). PKR-associated biological functions, such as inhibition of translation and cell apoptosis, have been demonstrated in association with the tumor suppressor functions of p53.…”