Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

Sky1: at the intersection of prion-like proteins and stress granule regulation.

Serine-arginine (SR) protein kinases regulate diverse cellular activities, including various steps in RNA maturation and transport. The yeast Saccharomyces cerevisiae expresses a single SR kinase, Sky1. Sky1 has a bipartite kinase domain, separated by an aggregation-prone prion-like domain (PrLD). The assembly of PrLDs is involved in the formation of various membraneless organelles, including stress granules; stress granules are reversible ribonucleoprotein assemblies that form in response to a variety of stresses. Here, we review a recent study suggesting that Sky1's PrLD promotes Sky1 recruitment to stress granules, and that Sky1 regulates stress granule dissolution by phosphorylating the RNA-shuttling protein Npl3.

The prion-like protein kinase Sky1 is required for efficient stress granule disassembly.

Stress granules are membraneless protein- and mRNA-rich organelles that form in response to perturbations in environmental conditions. Stress granule formation is reversible, and persistent stress granules have been implicated in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis. However, characterization of the factors involved in dissolving stress granules is incomplete. Many stress granule proteins contain prion-like domains (PrLDs), some of which have been linked to stress granule formation. Here, we demonstrate that the PrLD-containing yeast protein kinase Sky1 is a stress granule component. Sky1 is recruited to stress granules in part via its PrLD, and Sky1's kinase activity regulates timely stress granule disassembly during stress recovery. This effect is mediated by phosphorylation of the stress granule component Npl3. Sky1 can compensate for defects in chaperone-mediated stress granule disassembly and vice-versa, demonstrating that cells have multiple overlapping mechanisms for re-solubilizing stress granule components.

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains.

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN+], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

Distinct amino acid compositional requirements for formation and maintenance of the [PSI⁺] prion in yeast.

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.