# Mechanisms of Spliceosome Assembly and Regulation

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $372,895

## Abstract

PROJECT SUMMARY/ABSTRACT
RNA splicing is a key feature of human gene expression and a major contributor to expansion of genetic
information by alternative splicing. Splicing is carried out by a large and dynamic cellular machine called the
spliceosome. Spliceosomes are composed of small nuclear ribonucleoproteins (snRNPs) that assemble on
precursor transcripts (pre-mRNAs) to remove introns and splice together exons. This process must occur
precisely in order to preserve the genetic information carried in the mRNA. Critical for splicing is the correct
identification of the sites of RNA bond cleavage and formation [the 5' and 3' splice sites (SS) and the branch
site (BS)]. A number of different ATPases contribute to the fidelity of SS and BS recognition as well as carry
out extensive compositional and conformational remodeling of the spliceosome. Recently, biochemical studies
of splicing have been transformed by determination of dozens of different structures of yeast and human
spliceosomes by cryo-EM. Despite this structural revolution, much remains unknown about central features of
the splicing reaction. The goal of my laboratory’s research is to elucidate mechanisms of spliceosome
assembly and regulation in biochemical depth using a variety of techniques. We often use single molecule
fluorescence microscopy to deconvolute the complex and heterogeneous reaction pathways employed by the
splicing machinery. In recent work, we have studied mechanisms of 5'SS and BS recognition, assembly and
dynamics of the U6 snRNP, and developed methods for fluorescently-labeling, purifying, and inhibiting RNAs
and RNPs. Our vision for the next five years is to merge the insights obtained from structures of spliceosomes
with single molecule, biochemical, computational, and genetic experiments to address outstanding gaps in our
knowledge of splicing. These gaps include fundamental principles of RNP folding and assembly, the
mechanisms of regulated splicing, and the scarcity of specific and effective chemical inhibitors of the
spliceosome. As part of this vision, we will answer the following questions using multi-disciplinary approaches:
 1) How do RNA and protein co-fold to assemble the U6 snRNP?
 2) How is the spliceosome remodeled during creation of its active site?
 3) How do regulatory proteins promote splicing at weak 5'SS?
 4) How can we block ATPase-dependent transitions during splicing with small molecule inhibitors?
 5) How do we quantitatively analyze, compare, and integrate cryo-EM structures of spliceosomes?

## Key facts

- **NIH application ID:** 10133682
- **Project number:** 5R35GM136261-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Aaron Andrew Hoskins
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $372,895
- **Award type:** 5
- **Project period:** 2020-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133682

## Citation

> US National Institutes of Health, RePORTER application 10133682, Mechanisms of Spliceosome Assembly and Regulation (5R35GM136261-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10133682. Licensed CC0.

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