# Molecular Recognition During Pre-mRNA Splicing

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 2024 · $42,872

## Abstract

SUMMARY
The goal of our project is to understand the structural features guiding the initial stages of pre-mRNA splice site
recognition, which are critical for accurate pre-mRNA splicing and frequently dysregulated in human diseases.
We focus on the U2AF, SF1 and SF3B1 splicing factors directing the U2 small nuclear ribonucleoprotein (U2
snRNP) to the 3´ splice sites of pre-mRNAs. The U2AF subunits, U2AF2 and U2AF1, recognize the
polypyrimidine and AG-dinucleotide splice site signals. A third subunit, SF1 initially associates with the branch
point sequence of the pre-mRNA then is displaced by the SF3B1 subunit of the U2 snRNP. Dynamic
phosphorylation and dephosphorylation of SF3B1 is required for formation of the active spliceosome. Previously,
we made progress towards understanding the molecular underpinnings of the initial steps of 3´ splice site
selection. Using X-ray crystallography, biophysical techniques, and functional assays for pre-mRNA splicing in
human cells, we have shown that representative cancer-associated mutations at the U2AF2 – RNA interface
disrupt RNA binding and splicing. We have deciphered structural details showing how U2AF2 accommodates
diverse nucleotides of splice site signals. By complementary single molecule Förster resonance energy transfer
approaches, we further revealed that the U2AF2 conformation changes in response to different splice site
sequences as well as the U2AF1 subunit and its recurrent cancer-associated mutation. We have established
important, functional interfaces of U2AF2 with SF1 and SF3B1 during pre-mRNA splicing in human cells and
discovered that phosphorylation strongly reduces SF3B1 binding to U2AF2. However, these provocative results
raise new questions. First, what are the effects of recurrent U2AF2 mutations in neurodevelopmental disorders
compared to those in cancers? Second, how is dynamic SF3B1 phosphorylation and dephosphorylation
temporally regulated with U2AF2 dissociation prior to splicing? Third, how are the U2AF2, U2AF1, and SF1
subunits arranged to accurately recognize the splice site signals and ensure the fidelity of splicing? We address
these questions in the aims of this proposal by leveraging structural approaches (including X-ray crystallography,
cryoelectron microscopy, calorimetry, and fluorescence) and complementary functional assays (including co-
immunoprecipitations, pre-mRNA splicing assays, and transcriptome-wide sequencing). Altogether, the results
of these aims contribute to understanding the structural and functional underpinnings of 3´ splice site recognition
and its dysregulation in human disease.

## Key facts

- **NIH application ID:** 11035734
- **Project number:** 3R01GM070503-21S1
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** CLARA KIELKOPF
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $42,872
- **Award type:** 3
- **Project period:** 2004-07-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11035734

## Citation

> US National Institutes of Health, RePORTER application 11035734, Molecular Recognition During Pre-mRNA Splicing (3R01GM070503-21S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11035734. Licensed CC0.

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