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Understanding the role of prion-like domains in ribonucleoprotein granule dynamics

Date

2019

Authors

Boncella, Amy Elizabeth, author
Ross, Eric, advisor
Kennan, Alan, advisor
Peersen, Olve, committee member
Ackerson, Chris, committee member

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Abstract

Ribonucleoprotein (RNP) granules are membraneless organelles, comprised of RNA-binding proteins and RNA, that are integrally related with the cellular stress response. Stress granules and processing bodies (p-bodies) are the two primary types of RNP granules that reversibly assemble upon stress. Interestingly, many of the proteins that localize to stress granules and p-bodies contain aggregation-prone prion-like domains (PrLDs). Furthermore, mutations in the PrLDs of a number of stress granule-associated proteins have been linked to various neurodegenerative diseases, leading to the idea that aggregation-promoting mutations in these PrLDs cause stress granule persistence. Altogether, these finding suggest an important role for these domains in the dynamics of these assemblies. In order to gain a greater understanding of how PrLDs contribute to RNP granule biology, I have taken two different approaches. The first was to investigate how aggregation-promoting mutations affect stress granule and p-body dynamics. I introduced various aggregation-promoting mutations into the PrLDs of different stress granule and p-body proteins and assessed the ability of these granules to disassemble, hypothesizing that these mutations would cause RNP granule persistence, as is observed in disease. Interestingly, despite successfully increasing the aggregation propensity of these PrLDs, stress granules and p-bodies do not persist and can efficiently disassemble after stress relief. Given that aggregation-promoting mutations in PrLDs of RNP granule proteins fail to cause granule persistence, I took a second, less targeted approach towards understanding the roles of these domains in RNP granules. I focused on investigating how PrLDs are recruited to RNP granules by screening a set of PrLDs for ability to assemble into foci upon stress. Interestingly, many PrLDs are sufficient to assemble into foci upon various stresses, with robust recruitment to stress granules upon heat shock. Furthermore, several compositional biases are observed among PrLDs that are and are not sufficient to assemble upon stress. Using these biases, we have developed a reasonably accurate composition-based predictor of PrLD recruitment into heat shock-induced stress granules, which has been further validated using rational mutation strategies. This predictor is reasonably successful at predicting whether a PrLD will assemble into stress granules upon stress. Additionally, scrambling of PrLD sequences does not disrupt recruitment to stress granules. Together, these results suggest that PrLD localization to stress granules is based on composition rather than primary sequence.

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