RNA-seq analyses reveal the relevance of RNAs involved in ribosomal complex to induce mammalian prion protein aggregation and phase separation <i>in vitro</i>

Conformational conversion of cellular prion protein (PrP^C) into infectious PrP (PrP^Sc) is one of the most intriguing processes in modern Biology. It is well accepted that this transition is catalysed by one or more cofactors that lower the energy barrier between the different PrP forms. Among potential candidates, RNA molecules are strong contenders. Our group has pursued nucleic acids, both DNA and RNA, capable of inducing PrP misfolding, aggregation, and, more recently, phase separation, a process proposed to precede aggregation in degenerative disorders. We found that the interaction between recombinant PrP (rPrP) and total RNA extracted from neuroblastoma cells (N2aRNA) results in significant structural alterations. Here, we use rPrP:N2aRNA as a model to search for RNAs capable of inducing full-length murine rPrP phase separation and/or aggregation. N2aRNA was incubated with rPrP and after that, RNA-seq analysis was conducted with RNAs isolated from the insoluble material using two different protocols. We analysed thousands of RNA-seq reads, most of which represented ribosomal RNA molecules. The set of recovered molecules is heterogeneous; nevertheless, three low-complexity consensus motifs within the sequences of RNAs involved in ribosomal complex were identified as significantly enriched in the RNAs bound to rPrP, suggesting that a population of RNAs is responsible for inducing PrP phase transitions. We hypothesize that RNA transcripts enriched in a set of low complexity motif sequences with predicted structural similarities can be involved in PrP^C binding. This interaction would lead to phase separation and, ultimately, result in aggregation into scrapie-like species, in a stoichiometry-dependent manner.