Journal of Molecular Biology
Small, Highly Structured RNAs Participate in the Conversion of Human Recombinant PrPSen to PrPRes in Vitro
Introduction
Prion diseases represent a unique group of illnesses that are genetic, sporadic and infectious. An improperly folded form of the prion protein (PrPSc) is thought to be the causative agent of these diseases by some groups. This protein is involved in the pathogenesis of transmissible spongiform encephalopathies (TSE) such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cow and variant Creutzfeldt-Jakob disease in humans.1., 2., 3. The crucial pathogenic event in prion disease propagation is the structural conversion of benign, α-helices rich PrPC into the highly stable, β-sheet rich PrPSc isoform associated with infectivity.4 The PrPC→PrPSc conversion results in the alteration of some of the physical and biochemical traits of the protein such as a reduction in solubility and an increase in resistance to Proteinase K (PK) hydrolysis. Therefore, PrPC is often referred to as PrPSen as are recombinant forms of the protein. Conversion of PK sensitive PrP (PrPSen) into a PK resistant PrP isoform (PrPRes) is considered to be one of the indicators for transformation of PrPC into infectious PrPSc and, hence, disease progression. PrPSc isoforms aggregate into plaques, rods and scrapie-associated fibrils (SAF) that accumulate in the brains of affected animals and humans. Knockout mice lacking the prn-p gene, which encodes the PrP protein,5 are unable to contract prion disease after inoculation with infectious material containing PrPSc, demonstrating the requirement for both PrPC and PrPSc for development of prion disease.6 Mice inoculated with BSE extracts can develop prion disease in the absence of detectable PrPRes, suggesting that there may be other cellular components involved in PrPC→PrPSc conversion, PrPSc aggregation, prion disease transmission and propagation.7
Endogenous and extracellular nucleic acids have been considered as potential mediators in the modulation of PrP structure and prion disease development.8 This concept was developed based on data accumulated from in vitro experiments examining the interactions between PrP and DNA. These studies demonstrated that after 16 hours in non-physiological conditions (extremely high concentrations of PrP and DNA, 4 °C, pH 5), PrP binds to DNA and forms stable nucleoprotein complexes.9., 10., 11., 12. Because PrP in these complexes was partially resistant to PK proteolysis, it was proposed that DNA might promote β-sheet formation in PrPC.13 These nucleoprotein complexes aggregated into amyloid fibrils morphologically similar to PrPSc plaques, as shown by electron microscopy and Congo Red birefringence, despite the fact that PrPRes formation occurred under non-physiological conditions.14., 15. Recent in vitro conversion experiments show that although biochemically similar to PrPSc, all forms of PrPRes are not infectious, such as PrPRes isolated from the urine of infected animals.16
Recently, Zeiler et al.17 found that several small, highly structured RNA species (shsRNAs) derived from a collection of artificially constructed RNAs possess high affinity for the human recombinant prion protein (hrPrP) under physiological conditions (low concentrations, 37 °C, pH 7.5). The shsRNAs demonstrated a wide range of affinities for hrPrP, suggesting that RNA structure plays a role in the specificity and stability of the interaction.17 In this study, we further characterize the RNA-binding activities of PrP and examine, in depth, the effect of shsRNAs on inducing resistance of human recombinant PrPSen to PK digestion. We report here that interactions between shsRNAs and hrPrP in vitro under physiological conditions can lead to PrP aggregation and consequent conversion of PrPSen into PrPRes. In this study, we follow conversion solely by increased resistance of PrPSen to digestion by Proteinase K and not by techniques that resolve specific changes in structure (e.g. A-helix→B-sheet content). We also demonstrate that RNAs in these nucleoprotein complexes are protected against hydrolysis by ribonuclease A (RNase A). These results suggest potential molecular mechanisms of in vivo PrP conversion and a possible role of small RNAs in prion disease origin and progression.
Section snippets
Small, highly structured RNAs (shsRNAs) bind human recombinant prion protein (hrPrP) with high affinity and specificity
The binding activity of human recombinant PrP (hrPrP) for a number of shsRNAs was characterized by gel-shift assay. The RNAs were chosen on the basis of their ability to bind to hrPrP by filter-binding assay in buffer at physiological pH 7.5.17 The RNAs SC-2, and a variant containing a point mutation, SC-4, were added to this study because they were related to aptamers reported by Stefan Weiss (CEA) to specifically bind to PrPSc (reported at the PittCon 2001 Symposium, New Orleans, La, March
Discussion
Essential to understanding prion pathogenicity is the elucidation of factors that alter its structural features enabling the cellular prion protein to participate in the disease cascade. Due to the virus-like behavior of prion disease transmission and the co-purification of nucleic acids (NAs) with SAF, it has been argued that a nucleic acid may be the genetic element associated with TSEs.8., 23., 24., 25., 26., 27., 28. Recent work from several groups has demonstrated that NA binding alters
RNA preparation
The shsRNAs were synthesized by in vitro transcription with T7 RNA polymerase (Ambion, Epicentre) from DNA templates derived from sequenced plasmids. The RNAs were purified by organic extraction and precipitation, and then further purified by excision from denaturing polyacrylamide gels.
All of the RNAs used in this study (Table 1) have previously been shown to bind to hrPrP in simple buffer in the absence of BCS components.17 The origins and predicted secondary structures for MNV, MNVAP1,
Acknowledgements
We thank Dr Richard Carp and Dr Daryl Spinner (NYS Institute for Basic Research in Developmental Disorders, Staten Island, NY) for their valuable contributions and careful review and assistance in the preparation of the manuscript. We kindly give thanks to Psychogenics, Inc. (New York, NY) for providing mouse brains.
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