# Protein-driven dynamics of pre-mRNA splicing catalysis through single molecule microscopy

> **NIH NIH K99** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $100,000

## Abstract

Project Summary
 In eukaryotic organisms, transcribed RNA is processed from precursor messenger RNA (pre-mRNA) into
mature RNA in a process known as splicing. During this RNA processing mechanism, the non-coding regions of
pre-mRNA are removed, and the flanking regions are joined by a large molecular machine known as the
spliceosome. Spliceosomes do not exist pre-assembled into splicing active conformations. Instead, splice sites
(SS) are specifically chosen through the stepwise assembly of five small nuclear ribonuclear protein complexes
consisting of a small nuclear RNA and a large number of associated proteins. These spliceosome assemblies
are charged with correctly identifying and juxtaposing splice sites that are not explicitly sequence encoded in the
pre-mRNA. Adding to the complexity of splice site selection, >90-95% of human pre-mRNAs are alternatively
spliced by varying the configuration of which regions are joined and which are removed from multi-exon
containing genes. Splicing errors associated with alternative usage of splice sites are implicated in a large
number of human diseases such as Hutchinson-Gilford progeria syndrome (alternative 5'SS), dilated
cardiomyopathies (alternative 3'SS), Myelodysplastic syndromes (altered 3'SS preference) and early-onset
Parkinson Disease (cryptic splice site usage). Despite decades of research to characterize splicing mechanisms,
the mechanisms that control splice site usage are incompletely understood. To fill this knowledge gap, the long-
term goal of the candidate is to characterize the mechanisms that control splice site selection and the splicing
factors involved. In this project, I propose to investigate protein-driven RNA rearrangements during splicing
catalysis using single-molecule fluorescence microscopy methods through three specific aims. In aim 1, I will
implement a single molecule Förster resonance energy transfer (smFRET) approach to characterize a conserved
spliceosome rearrangement driven by the Prp22 helicase that leads to displacement of ligated mRNA from a
conserved region in the spliceosome catalytic core, U5 snRNA loop 1. A Prp22 variant will be used to stall
spliceosomes onto a surface immobilized pre-mRNA just after exon ligation but prior to release from the
spliceosome. Prp22-driven displacement of the ligated mRNA will subsequently be monitored using fluorescent
reporters installed on U5 snRNA loop 1 and the RNA substrate, respectively. Specific Aims 2 and 3 propose the
investigation of a human-specific protein, FAM32A, hypothesized to stabilize the interaction between the 5' exon
and U5 loop 1 in order to facilitate ligation to the 3' SS. Together, this work will answer questions about conserved
and metazoan-specific mechanisms involved in the late stages of pre-mRNA splicing catalysis. This project will
advance the applicant's career goal of running an independent laboratory at an academic institution in a way
that combines her graduate training in mechanistic enzymolog...

## Key facts

- **NIH application ID:** 10351379
- **Project number:** 1K99GM144735-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Elizabeth C Duran
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $100,000
- **Award type:** 1
- **Project period:** 2022-02-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10351379

## Citation

> US National Institutes of Health, RePORTER application 10351379, Protein-driven dynamics of pre-mRNA splicing catalysis through single molecule microscopy (1K99GM144735-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10351379. Licensed CC0.

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