Conversion of the cellular prion protein (PrP C ) into its altered conformation, PrPSc , is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ␤-sheet content and characteristics similar to those of PrP Sc . RNA molecules can also interact with PrP and potentially modulate PrP C to PrP Sc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP C 3 PrP Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.Prion diseases can be infectious, sporadic, or inherited (1). Regardless of their origin, they are related to modifications of a ubiquitous membrane-anchored protein, the prion protein (PrP).3 Through a poorly understood process, the cellular PrP isoform (PrP C ), an ␣-helix-rich protein, undergoes a profound conformational change, acquiring higher ␤-sheet content; the latter isoform is known as PrP Sc (Sc from scrapie) and is the only known component of the infectious prion particle (1-4).The protein-only hypothesis postulates that PrP Sc "multiplies" by catalyzing the conversion of PrP C into a likeness of itself, thus becoming responsible for its own propagation (5). This hypothesis is based strongly on the fact that PrP knock-out mice are resistant to prion infection, suggesting that endogenous PrP is necessary for prion propagation and infection (6). It was also suggested, however, that an additional unknown factor could influence the PrP C to PrP Sc conversion (7-10). This molecule would act by lowering the free energy barrier between PrP C and PrP Sc and triggering conversion (11,12). In this field, a great number of biological macromolecules have emerged as candidates for conversion catalysts. Cellular adhesion molecules, nucleic acids (NAs), basal membrane molecules, and sulfated glycans, among other biological macromolecules, have been reported to interact with PrP C and to induce its conversion in...

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP–DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP–D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP–nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP–nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.

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