Book of Abstracts: Albany 2007
June 19-23 2007
p53-RNA Interactions: in vitro, in Yeast, and in Human Cells
The tumor suppressor protein p53 is mutated in over half of human cancers. Despite more than 25 years of intense study, the complex regulation of this protein remains unclear. After serendipitously detecting RNA binding by p53 in the yeast three-hybrid system (Y3H), we explored the specificity and function of this interaction. The literature describing RNA-p53 interactions is controversial with reports of sequence-specific, sequence-nonspecific, and covalent interactions.
To elucidate the sequence specificity and possible function of the RNA-p53 interaction, we used the Y3H to screen and study p53-RNA binding in the context of the yeast nucleus. Data from electrophoretic mobility shift assays and circular dichroism spectroscopy of baculovirus-expressed p53 and synthetic peptides complement our in vivo studies of p53-RNA interactions. To further explore the possibility of RNA-p53 interactions in human cells, we used three techniques: co-immunoprecipitation, formaldehyde crosslinking immunoprecipitation, and UV crosslinking immunoprecipitation (CLIP).
Using random RNA libraries in a Y3H screen, we conclude that p53 is a sequence-nonspecific RNA binding protein. In vitro binding data further show that full-length p53 binds equally to all RNAs tested but poorly to tRNA. In general, RNA binding blocks sequence-specific DNA binding by p53. The C-terminus of p53 is necessary for RNA interaction in yeast and is both necessary and sufficient for a strong interaction in vitro. Murine and human C-terminal p53 peptides interact differently with RNA, and RNA is not bound by an acetylated form of the human p53 peptide. RNA does not appear to induce a structural change in the p53 C-terminal peptide, nor does the presence of C-terminal peptides affect the structure of RNA. We suggest that p53 is a sequence non-specific RNA binding protein, and p53-RNA interactions can be blocked by acetylation of the p53 C-terminus.
Although others have reported RNA co-immunoprecipitation with p53 from MCF-7 breast cancer cells expressing wild-type p53, we were unable to replicate this result. Contaminating RNA was co-immunoprecipitated from from several cell lines tested, regardless of p53 status. Furthermore, the majority of the p53 protein in MCF-7 and HCT-116 cells is acetylated and may, therefore, not bind to RNA. Thus, if p53-RNA interactions occur in human cells, it appears that the interaction is transient and involves only a minor fraction of the total p53 protein pool.
Department of Biochemistry and Molecular Biology