Small, Highly Structured RNAs Participate in the Conversion of Human Recombinant PrPSen to PrPRes in Vitro

https://doi.org/10.1016/S0022-2836(03)00919-7Get rights and content

Abstract

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrPC) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.

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.

References (37)

  • E. Derrington et al.

    PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1

    C. R. Acad. Sci.

    (2002)
  • G. Telling et al.

    Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein

    Cell

    (1995)
  • G.M. Shaked et al.

    Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity

    J. Biol. Chem.

    (1999)
  • S. Prusiner

    Novel proteinaceous infectious particles cause scrapie

    Science

    (1982)
  • T. Alper et al.

    Does the agent of scrapie replicate without nucleic acid?

    Nature

    (1967)
  • K. Pan et al.

    Conversion of alpha-helices into beta-sheets features in the formation of the scrapies prion protein

    Proc. Natl Acad. Sci. USA

    (1993)
  • S. Prusiner et al.

    Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies

    Proc. Natl Acad. Sci. USA

    (1993)
  • C. Lasmezas et al.

    Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein

    Science

    (1997)
  • Cited by (100)

    • A quantitative characterization of interaction between prion protein with nucleic acids

      2018, Biochemistry and Biophysics Reports
      Citation Excerpt :

      As mentioned above, small highly structured RNAs (shsRNA) bind to human recombinant prion protein with high affinity and specificity under physiological conditions demonstrated from gel electrophoresis studies [28]. These RNAs also can form highly stable nucleoprotein complexes with recombinant and cellular human prion protein (α-PrP) from various cell extracts and mammalian brain homogenates [28,42]. The human prion protein gene contains five copies of a 24 nt repeat that is highly conserved among species [43–45].

    • Neuronal Regulation of eIF2α Function in Health and Neurological Disorders

      2018, Trends in Molecular Medicine
      Citation Excerpt :

      PKR may also be activated by some RNA–protein complexes. For example, the conversion of cellular prion protein to the pathogenic form is enhanced by RNA [76,77], and prions can form RNA–protein aggregates [78,79] that are capable of activating PKR [80]. Similarly, the presence of large stress granules, which are sizable assemblies of non-translating mRNPs, can cause PKR activation [81].

    • Frameshifted prion proteins as pathological agents: Quantitative considerations

      2013, Journal of Theoretical Biology
      Citation Excerpt :

      The casual observation that a fraction of human PrP is associated with active ribosomes, especially in G2M phase cells, implicates PrP directly in the process of translation (Guichard et al., 2011). Normal-sequence PrPC has long been known to bind RNA molecules with diverse secondary and tertiary structures (Weiss et al., 1997; Adler et al., 2003; Sayer et al., 2004; Sekiya et al., 2006). More recent investigations have implicated the unstructured, octa-repeat-containing N-terminal region of PrPC in the process and a variety of functional effects resulting from RNA binding by PrP, including nucleic acid chaperoning, have been identified (Guichard et al., 2011; Gomes et al., 2008; Silva et al., 2008, 2010; Béland and Roucou, 2012; Cavaliere et al., 2013).

    View all citing articles on Scopus
    View full text