# Novel Tools to Investigate Local and Global RNA Conformations in the Spliceosome

> **NIH NIH R00** · UNIVERSITY OF OREGON · 2020 · $249,000

## Abstract

Project Summary
In eukaryotes, the vast majority of genes have their protein-coding regions (exons) split up, separated by
introns containing up to tens of thousands of nucleotides. The removal of introns, called “splicing”, is a critical
step in gene expression that allows for exquisitely fine-tuned regulation and, through alternative splicing,
diversifies a single gene into more than one protein. Splicing is executed by the spliceosome, a multi-
megaDalton macromolecular complex whose function requires interactions between the pre-messenger RNA
(pre-mRNA) substrate, five small nuclear ribonucleoprotein particles (snRNPs), and numerous additional
protein factors. Determining the roles of and interactions between these components is of central importance to
understanding the molecular mechanisms of the many human diseases in which aberrant splicing is
implicated.
 The recent application of single-molecule microscopy to the spliceosome has shed much light on the
molecular mechanism of splicing. However, the interactions between the snRNAs and the pre-mRNA have
remained difficult to probe due to the challenge of preparing snRNAs that are site-specifically fluorophore-
labeled. Furthermore, conformational changes can be tracked only on certain length scales, limited by the
sensitivity of the experimental techniques used, which are often based on Förster resonance energy transfer
(FRET). To address these challenges, Specific Aim 1 will study the rearrangement of interactions between U5
snRNA and the pre-mRNA in response to the action of RNA helicase Prp22. Site-specifically fluorophore-
labeled U5 will be prepared through by using a short peptide nucleic acid oligomer to stall transcription by T7
RNA polymerase at the desired labeling site, a general approach that avoids many of the downsides of other
RNA labeling methods. Specific Aim 2 proposes the novel technique of FRET-filtered spectroscopy (FFS),
which will utilize two closely spaced fluorophores as a FRET donor, and an additional fluorophore as an
acceptor. FFS will use electronic coupling between the two donors to reveal their local conformation as a
function of their distance from the acceptor, and can be expanded to utilize any type of fluorescence-detected
spectroscopy as a readout. This technique will be applied to Cy3- and Cy5-labeled RNA to study the unwinding
of RNA duplexes by Prp22. Specific Aim 3 combines the labeling method of Aim 1 with FFS, utilizing FRET-
filtered circular dichroism spectroscopy to determine the changes in local pre-mRNA conformation in the
vicinity of the branchpoint adenosine as purified Bact intermediates are chased through the first step of splicing.
This work will answer longstanding questions about the correlations between local and global RNA
conformations in the spliceosome, and involves novel methods that can be generalized to many different
biological systems. Aim 1 and the initial experiments for Aim 2 will be pursued in the laboratory of the
applican...

## Key facts

- **NIH application ID:** 9869899
- **Project number:** 5R00GM120457-04
- **Recipient organization:** UNIVERSITY OF OREGON
- **Principal Investigator:** Julia Reed Widom
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $249,000
- **Award type:** 5
- **Project period:** 2016-09-16 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9869899

## Citation

> US National Institutes of Health, RePORTER application 9869899, Novel Tools to Investigate Local and Global RNA Conformations in the Spliceosome (5R00GM120457-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9869899. Licensed CC0.

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