Biomedical Engineering ETDs

Publication Date

Summer 7-15-2020

Abstract

Intrinsically disordered proteins (IDPs) with RNA binding capability have recently been found to play important roles in several cellular processes, including the formation of phase separated membraneless organelles to regulate gene expression. An important class of membraneless organelles is stress granules, which enable cells to survive periods of environmental stress by regulating gene expression in the cytoplasm. Cells use a variety of ribonucleoprotein granules, primarily comprised of RNA and chaperone protein components, and can regulate cellular metabolism by sequestering and processing regulatory components, such as messenger RNAs, within these phases separated ‘organelles’. To recapitulate this cellular behavior, the genetically engineered model IDPs was used known as elastin-like polypeptides (ELPs) fused to RNA-binding peptide blocks derived from cellular RNA binding motifs. These modular IDPs are engineered to undergo a reversible phase transition. Below their transition temperature (Tt), ELP fusions are soluble in water, but at temperatures above their Tt, a phase transition occurs which leads to aggregation of RNA-ELP complexes. This programmable behavior demonstrates the utility of ELPs for the triggered formation of synthetic ribonucleoprotein membraneless organelles. The goal of this work is to provide a research basis for the overall engineering goal, which is to engineer a cleavable linker domain within the NA-binding blocks of the ELP fusions, where protease cleavage of previously concatenated blocks will trigger release of the RNA from the granules and the dissolution of the granule itself.

Language

English

Keywords

Elastin-Like Polypeptides, Intrinsically Disordered Proteins, Membraneless Organelles, Nucleic Acid binding, Synthetic Biology, Stress Granules, Phase Transition, Phase Behavior

Document Type

Thesis

Degree Name

Biomedical Engineering

Level of Degree

Masters

Department Name

Biomedical Engineering

First Committee Member (Chair)

Dr. Nick Carroll

Second Committee Member

Dr. Gabriel Lopez

Third Committee Member

Dr. Matthew Lakin

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