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

Award Number: R00GM144735
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 02/01/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2026

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Protein-driven dynamics of pre-mRNA splicing catalysis through single molecule microscopy - 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 enzymology with her ongoing postdoctoral training in RNA molecular biology and biophysics to characterize the mechanisms and assembly of complex macromolecular machines whose proper functions are vital to human health.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $249,000 )
2025	2025	UNIVERSITY OF ALABAMA AT BIRMINGHAM	701 S 20TH STREET	BIRMINGHAM	AL	35294	JEFFERSON	USA	Biomedical Research and Research Training	000	5	7/31/2025	NON-COMPETING CONTINUATION	$249,000
														Subtotal = $249,000

Issue Date FY: 2024 ( Subtotal = $273,165 )
2024	2024	UNIVERSITY OF ALABAMA AT BIRMINGHAM	701 S 20TH STREET	BIRMINGHAM	AL	35294	JEFFERSON	USA	Biomedical Research and Research Training	000	4	7/25/2024	NON-COMPETING CONTINUATION	$249,000
2024	2023	UNIVERSITY OF ALABAMA AT BIRMINGHAM	701 S 20TH STREET	BIRMINGHAM	AL	35294	JEFFERSON	USA	Biomedical Research and Research Training	001	3	7/25/2024	EXTENSION WITH OR WITHOUT FUNDS	$24,165
														Subtotal = $273,165

Issue Date FY: 2023 ( Subtotal = $249,000 )
2023	2023	UNIVERSITY OF ALABAMA AT BIRMINGHAM	701 S 20TH ST	BIRMINGHAM	AL	35294	JEFFERSON	USA	Biomedical Research and Research Training	000	3	8/25/2023	EXTENSION WITH OR WITHOUT FUNDS	$249,000
														Subtotal = $249,000

Grand Total All Awards = $771,165

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

Award Number: R00GM144735

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 02/01/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2026

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