Abstract
Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrPC into misfolded and aggregating PrPSc is the central event in prion disease, an alternative hypothesis suggests the generation of pathological PrPSc by rare translational frameshifting events in the octa-repeat domain of the PrP mRNA. Ribosomal frameshifting most commonly relies on a slippery site and an adjacent stable RNA structure to stall translating ribosome. Hence, it is crucial to unravel the secondary structure of the octa-repeat domain of PrP mRNA. Each of the five octa-repeats contains a motif (GGCGGUGGUGGCUGGG) which alone in vitro forms a G-quadruplex. Since the propensity of mRNA to form secondary structure depends on the sequence context, we set to determine the structure of the complete octa-repeat region. We assessed the structure of full-length octa-repeat domain of PrP mRNA using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy and selective 2′-hydroxyl acylation analysis by primer extension (SHAPE). Our data show that the PrP octa-repeat mRNA forms stable A-helical hairpins with no evidence of G-quadruplex structure even in the presence of G-quadruplex stabilizing agents.
Highlights
Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like CreutzfeldtJakob disease (CJD) and scrapie
Several controls verified that G-quadruplexes can form under the chosen experimental conditions, including those previously observed in isolated fragments of PrP mRNA (Supplementary Figs 2, 5–7)
Our results show salt-dependent oligomerization of the PrP octa-repeat mRNA, this was independent of KCl and PDS and occurred in the ΔG4 variant (Figs 1, 3)
Summary
Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like CreutzfeldtJakob disease (CJD) and scrapie. The octa-repeat region (amino acid residues 51–91) within the N-terminal domain of PrP appears crucial for aggregation[12]. This region contains a variable number of octa-repeats both within individuals of any mammalian species and between the predominant phenotypes from any species. The octa-repeat region of PrP mRNA is known to have a rich potential to form stem-loop[29], pseudoknot[30] or G-quadruplex[31] structures, all of which are associated with programmed frameshift events in other contexts[17], but there is a dearth of experimental evidence which would verify any role for these structures[32]. We hypothesized two alternative outcomes: 1) the accumulation of five G-quadruplexes within the octa-repeat domain would have a stacking effect[33,34] leading to potential stabilization of the G-quadruplexes; or 2) the flanking sequences would outcompete the G-quadruplex formation, resulting in the complete octa-repeat domain taking up an alternative structure
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