# Investigating the role of nuclear speckles in mRNP maturation

> **NIH NIH F32** · YALE UNIVERSITY · 2020 · $55,803

## Abstract

Project Summary/Abstract
 Mammalian nuclei are subdivided by a host of membrane-less organelles (MLOs) that separate from the
surrounding nucleoplasm to serve distinct functions. Nuclear speckles are among the most prominent MLOs in
mammalian nuclei, with 20-50 speckles per cell, each measuring ~1 µm in diameter. Speckles contain hundreds
of RNA binding proteins (RBPs) involved in several mRNP maturation events – including pre-mRNA splicing and
mRNP nuclear export – and also contain the highest concentration of post-transcriptional RNA in the nucleus.
Many components of speckles are oncogenes, and drugs that modulate speckles are anti-tumorigenic. Yet
despite their discovery over 50 years ago, the molecular function of speckles is unknown. The RNA within
speckles has not been defined, nor is it clear how speckles affect RNA processing or mRNP assembly. This
proposal employs novel strategies to elucidate the composition, regulation, and function of speckles, and will
directly test the hypothesis that speckles impact mRNP maturation and alter mammalian gene expression.
 The premise of this work is that investigating the function of speckles requires the study of entire speckles,
and not just the study of individual factors within them. Aim 1 will result in the development of a robust speckle-
purification method that uses fluorescent-particle sorting to isolate GFP-labeled speckles from cell lysate.
Quantitative proteomics and RNA-sequencing of purified speckles will provide the first comprehensive analysis
of their composition. Subsequent analysis will determine whether all or a subset of mammalian mRNPs traffic
through speckles, and will define whether RNAs are detained in speckles due to specific sequences and/or
incomplete splicing. Aim 2 will assess how speckles directly impact mRNP maturation. Two kinases that naturally
promote speckle disassembly in distinct physiological contexts will be separately hyperactivated or inhibited,
followed by genome-wide analysis of pre-mRNA splicing and nuclear export. Identifying common effects between
two kinases will reveal how speckle disassembly impacts mRNP maturation, as opposed to other effects of each
individual kinase. Aim 3 will define the RNA-protein interactions within speckles for essential RBPs. UV-
crosslinking followed by speckle-purification will identify speckle-specific RNA interactions for two SR proteins,
a family of RBPs that promote splicing and export of thousands of mRNAs. This analysis will reveal whether
RBPs within speckles bind to substrate RNAs that are unspliced, alternatively spliced, or consitutively spliced
isoforms. Ultimately, the methods and results stemming from this work will serve as a platform to investigate
speckles in the context of development and disease. The proposed research goals will provide significant training
in the use of many genome-wide approaches, including RNA-seq, proteomics, and eCLIP, as well as training in
modern genetic manipulations of human cells...

## Key facts

- **NIH application ID:** 9978571
- **Project number:** 5F32GM134598-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** David Phizicky
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $55,803
- **Award type:** 5
- **Project period:** 2019-07-01 → 2021-04-26

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9978571

## Citation

> US National Institutes of Health, RePORTER application 9978571, Investigating the role of nuclear speckles in mRNP maturation (5F32GM134598-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9978571. Licensed CC0.

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