# Molecular mechanism of ribosome biogenesis in the phase-separated nucleolus

> **NIH NIH F32** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2020 · $67,446

## Abstract

PROJECT/SUMMARY ABSTRACT
Ribosome biogenesis occurs in the nucleolus, a membrane-less organelle formed through liquid-liquid phase
separation (LLPS). However, little is known on how phase separation influences ribosome biogenesis and how
ribosomal RNA (rRNA) moves through the nucleolus. Recent work demonstrated rRNA and nucleophosmin
(NPM1) undergo LLPS, but the molecular determinants driving this interaction are not known. My preliminary
data indicates rRNA structure regulatesrRNA/NPM1 LLPS, suggesting a structure-based regulatory mechanism.
To confirm this, I will perform experiments to probe changes in rRNA conformation upon stabilization of rRNA
structure with Mg2+, and after LLPS with NPM1. I anticipate that NPM1 preferentially binds and stabilizes rRNA
in a structure that promotes ribosomal protein (r-protein) association. To bind and co-fold with rRNA, r-proteins
must enter the GC via a poorly understood mechanism, I propose that rRNA entry and its restructuring by NPM1
enhances r-protein entry. Once in the GC, r-protein/rRNA co-folding leads to abrogation of rRNA/NPM1 LLPS,
allowing rRNA to be released as a pre-ribosomal particle into the nucleoplasm. To test this, I will determine r-
protein partitioning into the GC and perform reconstitution studies using the E.Coli 30S small subunit. The studies
proposed above will provide insights into how rRNA escapes the GC, but not how rRNA moves through the
nucleolus. I propose the high rate of rDNA transcription leads to a flux of rRNA outward from the inner layer of
the nucleolus via a mass action mechanism. I will develop an in vitro system to examine if the outward flux of
rRNA and inward flux of r-proteins can recapitulate the early stages of ribosome biogenesis. In summary, the
proposed studies will provide novel insights into the role of phase separation in ribosome biogenesis and provide
the foundations for understanding how dysregulation of LLPS could lead to disease states such a Diamond-
Blackfan Anemia (a type of ribosomeopathy), and mutant NPM1-associated leukemias (e.g., acute myeloid
leukemia, acute promyelocytic leukemia, and anaplastic large cell lymphoma).

## Key facts

- **NIH application ID:** 10022121
- **Project number:** 5F32GM131524-02
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** Michele Tolbert
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $67,446
- **Award type:** 5
- **Project period:** 2019-09-17 → 2021-09-16

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10022121

## Citation

> US National Institutes of Health, RePORTER application 10022121, Molecular mechanism of ribosome biogenesis in the phase-separated nucleolus (5F32GM131524-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10022121. Licensed CC0.

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