Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.
The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.
Department of Biochemistry and Molecular Biology, Guggenheim 16, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota 55905, USA.
Kasandra J-L
KJ
Riley
Laura A
LA
Cassiday
Akash
A
Kumar
L James
LJ
Maher
D001483:Base Sequence; D015152:Blotting, Northern; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D016159:Tumor Suppressor Protein p53; D012313:RNA