# Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2023 · $7,712

## Abstract

PROJECT SUMMARY/ABSTRACT
This MIRA application is to support studies of how RNA binding proteins regulate choices in alternative splicing
and other posttranscriptional steps in mammalian gene expression. We will continue our studies of two families
of regulators, the Polypyrimidine Tract Binding Proteins and the Rbfox proteins. We will examine their
molecular mechanisms of action, their biological functions, and the roles of their extended regulatory programs
in neuronal development and mature neuronal function. Multiple human diseases, including several
neurodegenerative disorders, involve the dysfunction of RNA binding proteins and aberrant splicing regulation.
To develop treatments for such disorders, we need greater understanding of both the mechanisms and the
biology of alternative splicing. We will continue our studies of the nuclear and cytoplasmic Rbfox isoforms. We
will apply biochemistry and genome edited cell lines to examine how the eight RNA binding proteins of the
LASR complex bind with each other, and how the assembled complex interacts with nuclear Rbfox to regulate
splicing. RNAseq analyses of purified complexes and genomewide iCLIP analyses will map the binding of
LASR subunits relative to the known Rbfox binding sites to reveal how the RNA within the LASR complex is
organized. We will follow up on recent studies of cytoplasmic Rbfox isoforms to examine how these proteins
regulate the translation and stability of mRNAs encoding important synaptic proteins, such as Vamp1. We will
also continue our analyses of the Rbfox intrinsically disordered region and its ability to form molecular
condensates. These analyses will be extended to IDR’s in the LASR subunits to examine their homotypic and
heterotypic interactions, and the role of their condensation in splicing regulation. Our studies of the
mechanisms and biology of splicing repression by PTBP1 and PTBP2 will be continued. We will use
biochemical methods developed in earlier work and new mass spectrometry approaches to examine the
assembly and architecture of exon complexes repressed by PTBP1 and understand how PTBP1 blocks
productive spliceosome assembly. We will extend our investigation of the biological impact of two transitions in
neuronal splicing regulation: one induced early in neuronal development when PTBP1 is replaced with PTBP2,
and one occurring when PTBP2 is downregulated late in neuronal maturation. The roles of particular splicing
switches within the PTBP programs will be examined using stem cell differentiation protocols, CRISPR
mediated gene editing, and whole transcriptome expression and splicing analyses. Applying RNAseq to
subcellular fractions, we will characterize intron retention events controlled by PTBP1 and examine the
mechanisms that sequester RNAs on chromatin. Altogether these studies will yield new understanding of the
intricate molecular interactions that mediate the regulation of splicing and its misregulation in human disease.

## Key facts

- **NIH application ID:** 10797969
- **Project number:** 3R35GM136426-04S2
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Douglas L Black
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $7,712
- **Award type:** 3
- **Project period:** 2020-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10797969

## Citation

> US National Institutes of Health, RePORTER application 10797969, Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins (3R35GM136426-04S2). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10797969. Licensed CC0.

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