Date of Award

12-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Richard Kriwacki, Ph.D.

Committee

Tanja Mittag, Ph.D. Brenda Schulman, Ph.D. Susan Senogles, Ph.D. J. Paul Taylor, M.D., Ph.D.

Keywords

Membrane-less organelle, Nucleolus, Phase Separation, Structural Biology

Abstract

Nucleoli are nuclear membrane-less organelles that are the sites for ribosome biogenesis and serve as sensors of cellular stress. Weak, multivalent protein-protein interactions and interactions between disordered, low complexity domains (LCDs) and rRNA have been shown to promote liquid-liquid phase separation (LLPS) in vitro, suggesting a basis for the liquid-like behavior of nucleoli. Nucleophosmin (NPM1), a multifunctional and highly abundant nucleolar protein, exhibits structural features associated with LLPS suggesting a role in nucleolar organization. Specifically, NPM1 forms a pentamer through its N-terminal oligomerization domain and can bind to rRNA through its C-terminal nucleic acid binding domain. Multiple acidic tracts throughout NPM1, two within an intrinsically disordered region (IDR), confer additional multivalency and mediate interactions with proteins that contain multiple arginine-rich motifs (R-proteins). Using a variety of techniques, we have identified several nucleolar R-proteins which bind to and phase separate with NPM1. Here we show that the liquid-like properties of NPM1 droplets can be tuned by modulating the extent of electrostatic interactions within the droplet. We propose that a hierarchy of R-motifs, varying in valency and affinity, within nucleolar R-proteins exists which leads to a heterogeneous network of interactions between proteins and rRNA within nucleoli, thus promoting formation of a dynamic liquid-like phase conducive to ribosome biogenesis and other nucleolar functions.

ORCID

http://orcid.org/0000-0003-2911-5648

DOI

10.21007/etd.cghs.2016.0414

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