# Understanding Phase Separation in Biology and Disease

> **NIH NIH R35** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2021 · $538,500

## Abstract

Project Summary/Abstract
The process of liquid-liquid phase separation (LLPS) drives formation of numerous membrane-less organelles
in cells, the largest of which is the nucleolus. The nucleolus, through its multi-layered, dense liquid structure,
controls ribosome biogenesis and also serves as a center for cellular stress signaling involving the tumor
suppressors p53 and Arf. The nucleolus is a site of toxicity for di-peptide repeat (DPR) polypeptides observed
in the cells of amyotrophic lateral sclerosis (ALS) patients. In 2016 & 2018, we reported that the abundant
nucleolar protein, Nucleophosmin (NPM1), undergoes LLPS with ribosomal RNA (rRNA) and ribosomal
proteins (r-proteins), and with itself. We view NPM1 as a master organizer of the Granular Component (GC),
the outer region of the nucleolus, wherein rRNA assembles with r-proteins to form ribosomal subunits. Pre-
rRNA is transcribed in the center of the nucleolus and then captured through LLPS with the protein, Fibrillarin
(FIB1), in the Dense Fibrillar Component (DFC). After processing in the DFC, rRNA fluxes outwards and is
captured in the GC through LLPS with NPM1. Simultaneously, r-proteins are sequestered in the GC through
LLPS with NPM1. Our working model of ribosome assembly is that NPM1 “escorts” rRNA from the DFC into
the GC, where it encounters NPM1-escorted r-proteins moving oppositely. We propose that LLPS with NPM1
and FIB1 concentrates and co-localizes ribosomal components to assemble via a molecular hand-off model,
where NPM1 enhances rRNA:r-protein encounters, facilitating their binding, co-folding and ribosome subunit
assembly. Further, we propose that the Arf tumor suppressor functions within this dense liquid
microenvironment to independently modulate p53 activity and ribosome biogenesis, and that this
microenvironment is dramatically altered by the toxic DPRs observed in ALS.
We view LLPS-prone proteins through the lens of polymer theory, and apply our expertise with intrinsically
disordered proteins to discover the molecular mechanisms that enable nucleolar components (e.g., proteins
and RNA) to behave collectively through LLPS to form micron-scale, liquid nucleoli. We seek to understand
how the nature of protein and RNA inter-molecular interactions, often involving disordered and multivalent
protein regions, influences the material properties of the nucleolus and, consequently, ribosome assembly. The
emerging field of biological fluids requires new conceptual frameworks that blend the fields of structural and
cell biology with concepts from fluid physics and polymer theory. We will apply a wide-range of structural,
biophysical and biochemical, microrheology and cell biology techniques, to relate the molecular properties of
proteins and RNA to the material properties of phase separated bodies. Essentially, we seek to establish
disorder/multivalency-phase separation-function relationships, using the nucleolus as a model system.

## Key facts

- **NIH application ID:** 10139059
- **Project number:** 5R35GM131891-03
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** RICHARD W KRIWACKI
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $538,500
- **Award type:** 5
- **Project period:** 2019-07-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10139059

## Citation

> US National Institutes of Health, RePORTER application 10139059, Understanding Phase Separation in Biology and Disease (5R35GM131891-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10139059. Licensed CC0.

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