# Molecular Recognition During Pre-mRNA Splicing

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 2020 · $385,000

## Abstract

SUMMARY
Normal splicing of pre-mRNAs is a major source of eukaryotic transcript diversity, and defective pre-mRNA
splicing has emerged as a common hallmark of cancers and myeloid neoplasms. The goal of this proposal,
“Molecular Recognition in Pre-mRNA Splicing” is to understand regulation of the early stage of spliceosome
assembly, which is commonly affected among human diseases. In so doing, we seek to identify vulnerabilities
as potential therapeutic targets. Here, we focus on essential U2AF65 and SF3B1 splicing factors, which
together recruit U2 small nuclear ribonucleoproteins to the 3´ splice sites of pre-mRNAs. In the prior funding
period, we revealed that U2AF65 recognizes a nine-nucleotide polypyrimidine tract splice signal. We mapped
cancer-associated mutations on our U2AF65 structures and found clusters of U2AF65 hotspots at key interfaces.
In Aim 1 of the coming period, we will (1A) complete our views of U2AF65 recognizing different splice site
signals and (1B) test the hypothesized effects of cancer-associated U2AF65 mutations on 3´ splice site signal
recognition. The results of Aim 1 will aid algorithms to predict splice sites and the impacts of inherited splice
site mutations, and importantly establish the molecular consequences of acquired U2AF65 mutations in cancer.
In the prior funding period, we also showed that a U2AF65 paralogue, CAPERα, associates with the SF3B1
subunit both in human cell lysates and with purified proteins. In Aim 2 of the coming period, we will (2A) view
the SF3B1– U2AF65 interface and test its hypothesized relevance for pre-mRNA splicing in human cells. Then
we will (2B) investigate hypothesized phosphorylation-dependent regulation of SF3B1 associations with
U2AF65, CAPERα, and other alternative splicing factors during the splicing pathway. In support of the feasibility
of our aims, we have prepared diffracting crystals for structure determinations, methods for splicing factor co-
immunoprecipitation, knockdown, and re-expression, and we have identified U2AF65- and SF3B1-sensitive
splicing substrates as a basis to test our structure-guided hypotheses. SF3B1 is the most frequently mutated
splicing factor in human malignancies. Although SF3B1 hotspots are distinct from the U2AF65- and CAPERα-
binding sites, a “double whammy hit” that interferes with the functions of these collaborating factors, e.g. kinase
inhibitors such as already have been approved for cancer treatment, would selectively kill cancer cells carrying
SF3B1 mutations. Therefore it is important to understand the role of U2AF65 and the influence of
phosphorylation for SF3B1 functions. Altogether, the results of these aims will elucidate molecular mechanisms
of 3´ splice site recognition, and lay a foundation for future therapeutic strategies to treat cancers carrying
acquired splicing factor mutations.

## Key facts

- **NIH application ID:** 9978056
- **Project number:** 5R01GM070503-17
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** CLARA KIELKOPF
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $385,000
- **Award type:** 5
- **Project period:** 2004-07-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9978056

## Citation

> US National Institutes of Health, RePORTER application 9978056, Molecular Recognition During Pre-mRNA Splicing (5R01GM070503-17). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9978056. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*