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J. Biol. Chem., Vol. 283, Issue 28, 19616-19625, July 11, 2008

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Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells^*

Mariana P. B. Gomes, Thiago A. Millen, Priscila S. Ferreira, Narcisa L. Cunha e Silva, Tuane C. R. G. Vieira, Marcius S. Almeida, Jerson L. Silva¹, and Yraima Cordeiro^¶2

From the Programa de Biologia Estrutural, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Instituto de Biofísica Carlos Chagas Filho, and ^¶Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, 21491-590 Rio de Janeiro, Brazil

Conversion of the cellular prion protein (PrP^C) into its altered conformation, PrP^Sc, 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 β-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^CPrP^Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.

Received for publication, March 17, 2008 , and in revised form, May 1, 2008.

This work is dedicated to Leopoldo de Meis in commemoration of his 70th birthday.

^* This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, the Millenium Institute for Structural Biology in Biomedicine and Biotechnology (Conselho Nacional de Desenvolvimento Científico e Tecnológico Millenium Program), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (to J. L. S. and Y. C.), Financiadora de Estudos e Projetos of Brazil, an international grant from the International Centre for Genetic Engineering and Biotechnology (to J. L. S.), and a grant from L'Oréal, and Fundação Universitária José Bonifácio grant 13145-8 (to Y. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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The on-line version of this article (available at http://www.jbc.org) contains supplemental data, supplemental Table S1, and supplemental Figs. S1–S4.

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¹ To whom correspondence may be addressed: Instituto de Bioquímica Médica, BE, S10 21491-590, Rio de Janeiro, RJ, Brazil. Tel.: 55-21-2562-6756; Fax: 55-21-2561-2936; E-mail: jerson{at}bioqmed.ufrj.br. ² To whom correspondence may be addressed: Departamento de Fármacos, Faculdade de Farmácia, Bl. B Ss, S15, 21491-590, Rio de Janeiro, RJ, Brazil. Tel.: 55-21-2562-6756; Fax: 55-21-2561-2936; E-mail: yraima{at}pharma.ufrj.br.

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