# Mechanisms of Splice Site Selection in Health and Disease

> **NIH NIH R01** · UNIVERSITY OF ARIZONA · 2023 · $10,162

## Abstract

PROJECT SUMMARY
A key feature in pre-mRNA splicing is the processing step that pairs splice sites during the early stages of
spliceosome assembly. Yet, major gaps remain in the knowledge of specific molecular interactions that govern
splice site pairing, impeding understanding of mechanisms that regulate constitutive and alternative splicing.
Importantly, little is known as to how somatic mutations in splicing factors, including SF3A1, SRSF2, and
U2AF1, mediating key decisions in the early stages of spliceosome assembly produce myeloid malignancies.
The long-term goal of the proposed project is to determine fundamental mechanisms that maintain splicing
fidelity during the initial steps of spliceosome assembly and to identify molecular and cellular phenotypes
associated with splicing gene mutations that generate myelogenous blood diseases. The central hypothesis
is that interactions of SF3A1, a pivotal 3-splice site protein that bridges to its 5-splice site partner, U1 small
nuclear RNA (snRNA), plays crucial roles in splice site pairing and that mutations in SF3A1 disrupt these
functions. The central hypothesis is derived from preliminary data from the PI’s laboratory which reveal cross-
intron cooperation between SF3A1 and stem-loop 4 (SL4) of U1 snRNA in splice site pairing and novel
mediation of this interplay by RNA helicase UAP56. This hypothesis will be tested via 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) Elucidate the impact of SF3A1 mutations on
its splicing functions and perform a comparative analysis of the influence of mutations in SF3A1, U2AF1, and
SRSF2 on human hematopoietic stem and progenitor cells (HSPCs). Experiments in the first aim, will delineate
interactions between SF3A1 and UAP56 with U1 snRNA and other components of the splicing machinery via in
vitro splicing methods, and proximity-dependent biotin identification (BioID) technique. The action of SF3A1
and UAP56 on cellular mRNA profiles will be assessed by siRNA knockdown followed by RNA-seq.
Experiments in the second aim are designed to discover the consequences of SF3A1 mutations on its splicing
functions by in vitro splicing assays, and to identify mutation-induced splicing aberrations in human HSPCs by
RNA-seq. Hematopoietic ex vivo differentiation assays, coupled with immunophenotyping, will be employed to
identify abnormal phenotypic effects of SF3A1 mutations on human HSPCs. The strategy includes comparing
the influence of mutations in SF3A1 with those in SRSF2 and U2AF1 and is expected to reveal molecular and
cellular phenotypic defects that underlie abnormal hematopoiesis. Impact: Completion of the proposed work
will unravel the network of interactions between core spliceosomal components that govern commitment of an
intron to removal and reveal how splicing factor mutations impair splice site pairing and lead to spli...

## Key facts

- **NIH application ID:** 10808389
- **Project number:** 3R01GM127464-05S1
- **Recipient organization:** UNIVERSITY OF ARIZONA
- **Principal Investigator:** Shalini Sharma
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $10,162
- **Award type:** 3
- **Project period:** 2019-04-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10808389

## Citation

> US National Institutes of Health, RePORTER application 10808389, Mechanisms of Splice Site Selection in Health and Disease (3R01GM127464-05S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10808389. Licensed CC0.

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