Jan Bieschke

TATA-box binding protein associated factor 1 (TAF1) is the largest component of transcription factor IID (TFIID), a fundamental multiprotein complex for RNA polymerase II-mediated transcription. TAF1 is essential for promoter recognition, coactivator interaction, and normal development. Mutations in TAF1 cause developmental disorders and the lethal neurodegenerative disease X-linked dystonia-Parkinsonism (XDP). Our previous work suggested that this ∼170 kb gene has a more complex set of transcripts than currently catalogued. We therefore undertook a systematic assessment of Taf1 transcription in mouse, given its widespread use as a model organism and its high genetic homology with humans. Using targeted nanopore sequencing, we reveal extensive transcriptional diversity and differential abundance of Taf1 mRNAs across brain and body regions. We identify 19 novel transcript variants and multiple novel exons, and we predicted their protein domain architectures and modelled them using AlphaFold. Notably, we observed elevated RNA and protein expression in cerebellum compared to other brain regions. These findings substantially expand the transcriptional landscape of Taf1 and provide crucial insights to guide investigation of transcript-specific mechanisms in neurodevelopmental and neurodegenerative disorders, enabling the creation of more accurate disease models. Abstract Figure:

Prions replicate via the autocatalytic conversion of cellular prion protein (PrP^C) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt–Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation.

Prion diseases are a class of neurodegenerative diseases that are uniquely infectious. Whilst their general replication mechanism is well understood, the components required for the formation and propagation of highly infectious prions are poorly characterized. The protein-only hypothesis posits that the prion protein (PrP) is the only component of the prion; however, additional co-factors are required for its assembly into infectious prions. These can be provided by brain homogenate, but synthetic lipids and non-coding RNA have also been used in vitro. Here, we review a range of experimental approaches, which generate PrP amyloid assemblies de novo. These synthetic PrP assemblies share some, but not necessarily all, properties of genuine infectious prions. We will discuss the different experimental approaches, how a prion is defined, the non-protein requirements of a prion, and provide an overview of the current state of prion amplification and generation in vitro.

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). The pathway of PrP misfolding is still unclear, though previous data indicate the presence of a structural core in cellular PrP (PrP ^C ), whose cooperative unfolding presents a substantial energy barrier on the path to prion formation. PrP is a GPI-anchored membrane protein, and a number of studies suggest that membrane interactions play an important role in the conversion of PrP ^C to its disease-associated form, including a transmembrane form of PrP in which a highly conserved region (residues 110 - 136) spans the ER membrane. Insertion of this region results in the detachment of the PrP ^C first β-strand from the structural core. The effect of this removal on the structure, stability and self-association of the folded domain of PrP ^C is determined here through a biophysical characterisation of a truncated form of PrP ^C lacking this region. Whilst markedly destabilised, NMR chemical shifts show that the truncated protein exhibits tertiary structure characteristic of a fully folded protein and retains its native secondary structure elements, including the second strand of the PrP β-sheet, but with altered conformational flexibility in the β2-α2 loop and first α-helix. The latter is destabilised relative to the other helical regions of the protein, with markedly increased solvent exposure. This truncated form of PrP fibrilises more readily than the native form of the protein. These data suggest a stepwise mechanism, in which a destabilised “open” form of PrP ^C may be a key intermediate in the refolding to the fibrillar, pathogenic form of the protein.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrP^C) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, unphysiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening identified syntaxin 6 (Stx6) as a risk factor for Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed that Stx6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at a highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, Stx6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that PrP formed less-ordered aggregates with Stx6 instead of highly ordered fibrils, which contained Stx6. These data strongly suggest that the protein directly alters PrP self-assembly and, uniquely, acts as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation.

Summary: In prion diseases, fibrillar assemblies of misfolded prion protein (PrP) self-propagate by incorporating PrP monomers. Using total internal reflection and transient amyloid binding super-resolution microscopy, our study analyses elongation of single PrP fibrils to reveal polymorphic populations, featuring structural and dynamic heterogeneity similar to prion strains, which were previously hidden in ensemble measurements. PrP fibrils elongated along a preferred direction by an intermittent ‘stop- and-go’ mechanism. Fibrils fell into three main populations, which each displayed distinct elongation mechanisms incorporating different monomer structures and which maintained their properties even under elongation conditions favouring a different fibril type. Elongation of RML and ME7 prion rods likewise exhibited unique kinetic features. The discovery of polymorphic fibril populations of amyloid and prions growing in competition suggests that prions may present as quasispecies of structural isomorphs and that the replication environment may tilt the balance between prion isomorphs and amyloid species. Highlights: Synthetic prion fibril populations contain structurally distinct fibril types Fibril types faithfully elongate by different mechanisms Fibril types compete for substrate depending on environment Fibril populations model quasi-species behavior of prion strains eTOC: Replication of different prion strains causes distinct disease phenotypes. Sun et al. analyzed the growth of individual synthetic prion protein fibrils by super-resolution microscopy and found populations of structurally distinct fibril types, which grew in competition to each other as a quasi-species, recapitulating basic prion strain characteristics in vitro .