SEQUENCE FEATURES THAT TARGET PRION-LIKE PROTEINS TO MEMBRANELESS ORGANELLES

Abstract

Membraneless organelles (MLOs), such as stress granules (SGs) and processing bodies (P-bodies), form in response to cellular stress and play a critical role in the cell’s ability to withstand adverse conditions. SGs and P-bodies are involved in post-transcriptional regulation and assemble through the condensation of non-translating mRNAs and RNA-binding proteins. Many RNA-binding proteins within MLOs contain prion-like domains (PrLDs), which are thought play a role in MLO assembly. Mutations within PrLDs associated with SGs have been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). While mechanisms of MLO assembly remain unclear, understanding the mechanisms governing MLO assembly and their specificity within the cellular environment could provide insights into the regulation process of their assembly and disassembly. Defining the role of PrLDs in MLO assembly could provide key insights into the regulation of their formation and disassembly.In the work presented in this dissertation, I investigated the sequence features that target PrLDs to SGs, using yeast SGs as a model system alongside synthetic PrLDs and fluorescence microscopy to determine how individual amino acid residues influence PrLD recruitment. In Chapter 2, rational mutagenesis was employed to quantitatively assess the contribution of individual amino acids to synthetic PrLD recruitment to SGs, revealing that highly hydrophobic and aromatic residues enhanced PrLD localization to SGs, while prolines reduced it. In Chapter 3, the interplay between segments within PrLDs that positively or negatively influence their partitioning to SGs was examined, with findings suggesting that the presence of a positive SG-prone segment exerts a dominant effect in localizing fused PrLDs to SGs. Finally, in Chapter 4, specificity of targeting between different condensate types was investigated by determining whether sequence features driving SG localization also influence PrLD recruitment to P-bodies. These findings revealed that PrLDs alone are generally insufficient for P-body targeting, and that additional domains, including RNA-binding domains and, in some cases, dimerization domains, contribute to P-body localization. Collectively, these findings advance our understanding of the molecular mechanisms underlying MLO assembly, condensate specificity, their roles in cellular stress responses.