# Comprehensive Maps of U1 snRNP Binding to Nascent RNA in Human Cells

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2022 · $429,000

## Abstract

PROJECT SUMMARY/ABSTRACT
We propose to develop new methods for the global identification of protein and RNP interactions with nascent
unspliced RNA. We will use these methods to produce comprehensive maps of spliceosomal U1 snRNP binding
across the human transcriptome. U1 functions in pre-mRNA splicing, in the suppression of premature
cleavage/polyadenylation, and in the nuclear retention of RNA. However, information on its binding sites is very
limited, and how these sites differ for the different functions of U1 is not understood. Unspliced introns in nascent
RNA fractionate with the chromatin, and we recently showed that within the chromatin compartment, splicing
factors engage in interactions not seen elsewhere. We developed methods to isolate proteins and RNP’s that
allowed identification of new regulatory proteins interacting with the U2 snRNP, and the isolation of U2-bound
pre-mRNA fragments encompassing the intronic branch points of HEK293 cells. We call this method
fractionation/immunopurification/RNAse protection (FIRP) and find the FIRP maps of U2/pre-mRNA interactions
to be more comprehensive than previous approaches for mapping branchpoints. We now propose to adapt FIRP
to characterizing interactions of the U1 snRNP with pre-mRNA. We will optimize the extraction of material from
chromatin to obtain U1 snRNP complexes bound to pre-mRNA. We will develop new anti-U1 antibodies that
allow 5’ splice site binding analysis across all types of cells. These methods will be applied both to FIRP assays
of U1 snRNP binding and to iCLIP analyses of U1 protein contacts on chromatin-associated RNA. By
characterizing U1 binding sites on a global scale and developing methods for assaying its interactions in different
cells, regulatory environments and genetic backgrounds, we can examine the processes of 5’ splice site
recognition with new breadth and precision.

## Key facts

- **NIH application ID:** 10507429
- **Project number:** 1R21HG012624-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Douglas L Black
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $429,000
- **Award type:** 1
- **Project period:** 2022-08-12 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10507429

## Citation

> US National Institutes of Health, RePORTER application 10507429, Comprehensive Maps of U1 snRNP Binding to Nascent RNA in Human Cells (1R21HG012624-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10507429. Licensed CC0.

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