Tumor suppressor p53 is a well-characterized transcription element that binds DNA. interactions between p53 and the 5-UTR of its own mRNA transcript [16], and others suggested that p53 can bind the 5′-UTR of Cdk4 mRNA [17]. Similarly, Galy et al. reported that recombinant p53 can bind the 5-UTR of the FGF-2 mRNA [18]. In contrast, reports of p53 RNA-RNA annealing activity [13] and sequence-nonspecific RNA binding [4, 6, 7, 19] suggested that p53 binds RNA promiscuously. Acetylation of four p53 C-terminal lysine residues eliminates detectable RNA binding [7], as it does for sequence-nonspecific DNA binding [8]. We wished to test the hypothesis that p53 associates with specific target RNAs in living cells. A AZD-3965 kinase activity assay prior study of p53-RNA interactions in human cells suggested that RNA is co-immunoprecipitated with p53 using Do-1 and Do-7 antibodies, which recognize N-terminal p53 epitopes (Fig. 1A,B; [19]). Recovered RNAs were recognized by radiolabeling but weren’t cloned [19]. A drawback of such RNA co-immunoprecipitations may be the potential redistribution of complexes after cell lysis [20]. Nonspecific RNA contamination of immunoprecipitations is definitely a problem also. High-salt washes might enhance stringency but might disrupt both particular and nonspecific RNA-protein interactions [7]. One method of increase stringency requires covalent cross-linking having a chemical substance agent such as for example formaldehyde, which catches protein-RNA interactions quickly and reversibly (Fig. 1B, middle; [21]). The UV-cross-linking and immunoprecipitation (CLIP) process was lately reported to facilitate the recognition and cloning of protein-bound RNAs (Fig. 1B, correct; [22]). CLIP requires the short UV irradiation of live cells accompanied by extract planning for immunoprecipitation (Fig. 1B, correct; [22]). Selection among the countless commercial antibodies particular for p53 (Fig. 1A) can enhance the stringency and specificity of the technique. The lysate useful for immunoprecipitation can be treated with nucleases to remove AZD-3965 kinase activity assay DNA contamination, producing protein-bound RNA tags. Linker oligonucleotides are ligated towards the radiolabeled RNA tags, as well as the protein-RNA complexes are separated by SDS-PAGE. RNA tags are purified, amplified, cloned, and sequenced (Fig. 1B, correct). Our earlier research of RNA binding by p53 in candida and proven that p53 with imperfect post-translational adjustments binds RNA without obvious series- or structure-specificity [4, 6, 7]. It’s possible a partially-modified type of p53 could can be found with a number of specific RNA companions. RNA co-immunoprecipitation with p53 was carried out to test the hypothesis that RNA binding partners for p53 AZD-3965 kinase activity assay can be cloned from human cells. Experimental Procedures Cell culture and antibodies MCF-7 (+/+) human breast cancer cells were grown in Dulbecco’s modified Eagle’s medium, PC-3 (?/?) human prostate cancer cells were grown in McCoys 5a medium, and HCT116 human colorectal carcinoma AZD-3965 kinase activity assay cells (both p53 WT and ?/?; [23]) were grown in RPMI medium. Media were supplemented with 10% fetal bovine serum. Anti-p53 PAb 1801 and PAb 421, and anti-c-myc Ab1 antibodies were purchased from EMD Biosciences, anti-HA HA.11 antibody from Covance, anti-p53 (PAb 246, C-19, Do-1, HRP-conjugated Do-1, and Do-7) antibodies from Santa Cruz Biotechnology, 2Ac (p53 Ac373/Ac382) from Upstate, and anti-p53 Do-12 from Chemicon International. RNA co-immunoprecipitation Untreated cells (MCF-7, PC-3, HCT116, and HCT116 +/+) (Fig. 2A, lane 2) but not ?/? HCT116 cells (Fig. 2A, lane 1). p53 was detected in MCF-7 cells (Fig. 2A, lane 4), but not in PC-3 cells (Fig. 2A, lane 3). p53 proteins in MCF-7 and HCT116 cell Rabbit Polyclonal to E2F4 lines displayed slightly different electrophoretic mobilities, suggesting differences in post-translational modifications (Fig. 2A, compare lanes 2 and 4). p53 was immunoprecipitated with Do-7 antibody from AZD-3965 kinase activity assay HCT116 (+/+) and MCF-7 cells (Fig. 2A, lanes 6 and 8) but not from HCT116 cells (?/?) or PC-3 cells (Fig. 2A, lanes 5 and 7). Open in a separate window Fig. 2 RNA is purified from cell lysates.