Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

Abstract

Background:The mechanisms of prion strain mutation and its dependence on the environment are not known. Results: Glycosylation status of PrP C substrate and cofactors controls the selectivity of amplification of classical and atypical PrP Sc . Conclusion: Amplification selectivity of alternative prion states can be regulated by modification of a substrate and cofactors in PMCA. Significance: Alternative prion states can be selectively amplified from a mixture.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

“…The mammalian homologue of VIR was recently shown to associate with m 6 A writer proteins (Ortega et al ., 2003; Horiuchi et al ., 2013; Schwartz et al ., 2014), we therefore decided to test experimentally whether a similar association also existed in Arabidopsis . We used tandem affinity purification (TAP) followed by proteolysis and mass spectrometry (Van Leene et al ., 2011) to identify proteins that associated with the VIR‐GS bait in Arabidopsis suspension cell cultures.…”

“…Using this approach, we consistently identified FIP37 (Zhong et al ., 2008) and HAKAI (Fig. S3; Fujita et al ., 2002; Horiuchi et al ., 2013) co‐purifying with VIR and MTB (Bujnicki et al ., 2002) also significantly enriched with a lower confidence score (Fig. 2a; Table S7).…”

“…In D. melanogaster , the FIP37 homologue Fl(2)D closely associates with VIRILIZER (Ortega et al ., 2003) and interacts genetically with methylation pathways to regulate sex‐specific splicing events (Haussmann et al ., 2016; Lence et al ., 2016). In human cells their orthologues WTAP and KIAA1429 also co‐purify (Horiuchi et al ., 2013; Schwartz et al ., 2014). Our data thus indicate that writer proteins in Arabidopsis more closely resemble those in mammals than yeast.…”

“…More recently, it has also been implicated with influencing RNA–protein interactions in animal systems (Fujita et al ., 2002; Figueroa et al ., 2009) and has been reported in association with m 6 A writers (Horiuchi et al ., 2013), but a role in m 6 A writing has not been proposed until now. Although believed to act on E‐cadherin complex at the plasma membrane, human and D. melanogaster HAKAIs are localized predominantly in the nucleus and in lower amounts at the plasma membrane and in the cytoplasm (Figueroa et al ., 2009; Kaido et al ., 2009).…”

“…HAKAI was first characterized in humans as a RING domain E3 ubiquitin‐ligase that mediates the post‐translational downregulation of E‐cadherin at the plasma membrane (Fujita et al ., 2002). It has recently appeared in animal proteomics interaction lists that include other m 6 A writer complex members (Horiuchi et al ., 2013); however, until now, a role in m 6 A writing has not been proposed. The demonstration here – that this interaction is conserved across kingdoms and that plant HAKAI is functionally required for full mRNA methylation – may indicate that a similar role for mammalian HAKAI should also be considered.…”