Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.
Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.
D000260:Adenoviruses, Human / Q000235:genetics* / Q000528:radiation effects; D002472:Cell Transformation, Viral; D006367:HeLa Cells / Q000528:radiation effects; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins / Q000235:genetics*; D011485:Protein Binding; D012313:RNA / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics*; D012342:RNA, Small Nuclear; D012367:RNA, Viral / Q000235:genetics*; D006163:Ribonuclease T1; D012261:Ribonucleoproteins / Q000235:genetics*; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000235:genetics*
D009363:Neoplasm Proteins; D009698:Nucleoproteins; D012334:RNA, Neoplasm; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins; D012313:RNA; D006163:Ribonuclease T1