# Comprehensive Maps of Early Spliceosome and Regulator Binding to Nascent RNA in Human Cells

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2024 · $639,134

## Abstract

PROJECT SUMMARY/ABSTRACT
We propose to develop new methods for the global identification of protein and RNP interactions with nascent
unspliced RNA in human cells. We will use these methods to produce comprehensive binding site maps for
several important molecules in splicing. In previous work, we isolated proteins and RNP’s from the chromatin
fraction of cells to identify a new complex of splicing regulatory proteins, Rbfox/LASR, as well as a new U2
snRNP complex containing novel regulatory proteins. We further found that sequencing the pre-mRNA
fragments bound within both these complexes allows the comprehensive mapping of their transcriptome-wide
interactions. This “IPseq” mapping of U2/pre-mRNA interactions provides sensitive detection of intron
branchpoints across the expressed transcriptome. We now propose to apply IPseq to develop binding maps of
several key splicing components. We will continue ongoing studies of Rbfox/LASR and the U1 snRNP, and we
will apply the method to the early spliceosome component: U2AF2. In preliminary data, we find that IPseq
generates precise maps of the binding of both Rbfox1 and the LASR subunits on pre-mRNAs. Analyses of
these data will yield important understanding of how the large Rbfox/LASR complex interacts with RNA, and
how individual bound elements cooperate to generate a splicing regulatory code. The U1 snRNP recognizes
the 5’ splice during early spliceosome assembly, binds within splicing silencer elements, and also acts to
suppress premature cleavage/polyadenylation events in nascent RNA transcripts. Genomewide information on
U1 snRNP binding is very limited, and the global analysis of its many RNA interactions will provide new
insights into its multitude of functions. The U2AF heterodimer binds the 3’ splice site during early spliceosome
assembly and is essential for the formation of an exon definition complex. A combined map of U1 snRNP and
U2AF binding sites will allow their assessment as parts of possible cryptic exons and allow better prediction of
mutational activation of cryptic splicing. By building a comprehensive map of the key factors for exon definition,
our goal is to enable better prediction of pathogenic mutations affecting splicing. Finally, we believe that
chromatin RNA IPseq can be broadly applied to many other cellular components.

## Key facts

- **NIH application ID:** 10943551
- **Project number:** 1R01HG013670-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Douglas L Black
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $639,134
- **Award type:** 1
- **Project period:** 2024-09-17 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10943551

## Citation

> US National Institutes of Health, RePORTER application 10943551, Comprehensive Maps of Early Spliceosome and Regulator Binding to Nascent RNA in Human Cells (1R01HG013670-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10943551. Licensed CC0.

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