# Mechanisms of Splice Site Selection in Health and Disease

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2023 · $118,292

## Abstract

PROJECT SUMMARY
In the last decade, significant progress has been made in the understanding of core components involved in
splicing catalysis in mature spliceosome. However, major gaps remain in the knowledge of interactions that bring
intron ends together to pair splice sites in the early steps of spliceosome assembly. Continued lack of this
knowledge is a significant impediment to an improved understanding of mechanisms that regulate constitutive
and alternative splicing to shape the cellular transcriptome and how somatic mutations in splicing factors involved
in these early steps cause pathogenesis in myeloid malignancies. Our long-term goal is to determine
fundamental mechanisms in maintenance of splicing fidelity in the early steps of spliceosome assembly and
identify molecular and cellular phenotypes associated with mutations in splicing genes in myeloid diseases. Our
central hypothesis is that interactions of SF3A1 with its partner, the U1 small nuclear RNA (snRNA), have crucial
functions in splice site pairing and that mutations in SF3A1 disrupt these functions. This hypothesis has been
formulated on the basis of our preliminary data demonstrating the role of the cross-intron interaction between
SF3A1 and the stem-loop 4 (SL4) of the U1 snRNA in splice site pairing and potential mediation of this interaction
by the RNA helicase UAP56. We plan to test our central hypothesis through two specific aims: 1) Determine the
molecular mechanism(s) whereby SF3A1-dependent splice site pairing events contribute to spliceosome fidelity
and generate normal mRNA profiles, and 2) Determine the impact of SF3A1 mutations on its splicing functions
and perform comparative analysis of effects of mutations in SF3A1, U2AF1, and SRSF2 on human hematopoietic
stem and progenitor cells. In the first aim, we will delineate the interactions of SF3A1 and UAP56 with U1 snRNA
and other components of the splicing machinery by in vitro reconstituted splicing methods and the proximity-
dependent biotin identification (BioID) technique. We will determine the impact of SF3A1 and UAP56 on cellular
mRNA profiles by siRNA knockdown followed by RNA-seq. In the second aim, we will determine the
consequences of SF3A1 mutations on its splicing functions by in vitro reconstituted splicing assays and identify
mutation-induced splicing aberrations in human HSPCs by RNA-seq. We will employ ex vivo hematopoietic
differentiation assays to identify the abnormal phenotypic effects of SF3A1 mutations on human HSPCs by
immunophenotyping. Our strategy includes comparing the influence of myeloid disease mutations in SF3A1 with
those in SRSF2 and U2AF1 and is expected to reveal molecular and cellular phenotypic defects that underlie
myeloid disease pathogenesis. Completion of the proposed research will advance our understanding of
interactions between core spliceosomal components that govern the commitment of an intron to removal and
how splicing factor mutations impair splice site pairing lead...

## Key facts

- **NIH application ID:** 10797554
- **Project number:** 3R01GM127464-05S3
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Shalini Sharma
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $118,292
- **Award type:** 3
- **Project period:** 2023-03-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10797554

## Citation

> US National Institutes of Health, RePORTER application 10797554, Mechanisms of Splice Site Selection in Health and Disease (3R01GM127464-05S3). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10797554. Licensed CC0.

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