ASSOCIATION OF SCRAPIE PRION PROTEIN AND PRION PROTEIN-RNA STEM-LOOP WITH NUCLEAR CARBOHYDRATE-BINDING PROTEIN-35 AND OTHER RNA-BINDING PROTEINS

A number of cellular proteins were identified that bind to the predict ed RNA stem-loop structure of prion protein (PrP) RNA; a virtually ide ntical set of RNA-binding proteins was found to associate with the tra ns-activating region TAR of the human immunodeficiency virus-1. The pr edicted hairpin elements of the PrP mRNA contain, like TAR RNA, a CUGG G sequence in the loop and a uridine and adenine bulge in the stem; th ese features are unique among cellular RNAs. UV cross-linking of RNA.p rotein complexes formed between PrP RNA and HeLa nuclear protein yield ed four prominent RNase-resistant complexes, in addition to some minor bands, which migrated at approximate to 90, 68, 42, and 37kDa under d enaturing conditions. The presence of multiple PrP RNA-binding, as wel l as TAR RNA-binding polypeptides was also demonstrated in Northwester n assays with nuclear extracts from mouse ascites, liver, and spleen, whereas only one PrP RNA-binding protein (a doublet with an approximat e molecular mass of 35kDa) was found in brain extract from rat. The nu clear beta-galactoside-specific lectin, CBP35 (carbohydrate-binding pr otein with a molecular mass of 35 kDa), which has been identified in n uclear ribonucleoprotein (RNP) complexes from a variety of mammalian t issues and cells, was among those proteins which bind to PrP RNA. The cellular prion protein, PrPC, was found to be unable to bind PrP RNA d irectly; however, this protein could be detected in the RNP/CBP35 comp lex formed between PrP RNA and rat brain extracts. Association of PrPC with RNP/CBP35 complex was abolished by RNase treatment. CBP35 could be also detected in purified infectious scrapie prions, suggesting a p ossible role in prion replication.