# The central roles of SRSF1 in early-stage spliceosome assembly

> **NIH NIH R35** · UNIVERSITY OF ALABAMA AT BIRMINGHAM · 2023 · $371,250

## Abstract

Project Summary:
Alternative splicing processes over 95% of human mRNA and enables a single gene to encode
distinct protein isoforms of different functions. Dysregulation of alternative splicing causes
incorrect selection of exons and consequently various human diseases. Alternative exons are
selected in early-stage spliceosome assembly. Due to our limited knowledge about early-stage
spliceosome assembly, it is still challenging to develop therapies for diseases related to
aberrant RNA splicing. Early-stage spliceosome assembly involves selection of exons and
recruitment to the splicing sites of ribonucleoprotein complexes U1 and U2. These processes
depend on the interplay of Ser/Arg-rich proteins (SR), U1-70K and U2AF-35. SR proteins are
the key factors that coordinate all these events. The SR family consists of 12 members and
shares Arg-Ser repetitive regions (RS) that are subjected to phosphorylation. In this proposal,
we have selected the prototype of the family, SRSF1, as a model to investigate the central roles
of SR proteins in spliceosome assembly. Mounting cellular studies have shown that SRSF1
promotes inclusion of exons by binding to exonic splicing enhancer RNA motifs, and
phosphorylation of SRSF1 regulates not only the overall splicing pattern, but also the
spliceosome assembly. Despite the progress in cellular studies, elucidating the mechanisms by
which phosphorylation of SRSF1 regulates exon selection and spliceosome assembly is
challenging due to low solubility of SR proteins, U1-70K and U2AF-35. Our lab has obtained all
three of these proteins in the soluble full-length form. With this success, we have found that the
SRSF1 RS region (a) displays RNA-binding preference and its phosphorylation inhibits RNA
binding; (b) is essential for interaction with U1-70K and U2AF-35, which are responsible for
recruitment of U1 and U2 complexes, respectively; (c) mediates phase separation, which is
consistent with its role in organizing nuclear speckles. This proposal will (1) use the high-
throughput method RNA Bind-n-Seq to systematically investigate how phosphorylation
regulates RNA-binding specificity of SRSF1; (2) use a combination of NMR, molecular dynamic
simulations and other biophysical methods to elucidate the structural mechanism by which
SRSF1 interacts with U1-70K and U2AF-35; (3) investigate how the RS region balance its roles
in modulating RNA-binding affinity, mediating protein interactions, and organizing phase
separation in the phase-separated state. In summary, our proposal will advance our knowledge
of exon selection and splicing factor interaction during early-stage spliceosome assembly.

## Key facts

- **NIH application ID:** 10678784
- **Project number:** 5R35GM147091-02
- **Recipient organization:** UNIVERSITY OF ALABAMA AT BIRMINGHAM
- **Principal Investigator:** Jun Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $371,250
- **Award type:** 5
- **Project period:** 2022-08-08 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10678784

## Citation

> US National Institutes of Health, RePORTER application 10678784, The central roles of SRSF1 in early-stage spliceosome assembly (5R35GM147091-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10678784. Licensed CC0.

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