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Role of pseudouridines in pre-mRNA processing

Award Number: R00GM135537
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: 08/01/2020
PERIOD OF PERFORMANCE END DATE: 01/31/2025

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Role of pseudouridines in pre-mRNA processing - PROJECT SUMMARY Recent development of transcriptome-wide approaches to detect modified nucleotides has revealed an expanding repertoire of modified nucleotides in messenger RNA. RNA modifications represent a new layer of eukaryotic gene regulation. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated in different conditions. However, the endogenous functions of Ψ in mRNA are not currently understood. We have shown that nascent pre-mRNA is pseudouridylated co-transcriptionally at thousands of locations by multiple Pseudouridine Synthases (PUSs). We found that pre-mRNA Ψs are enriched in regions important for splicing regulation and overlap splicing factor binding sites. Genetic manipulation of Ψ synthases leads to widespread alternative splicing and individual Ψ directly affect splicing in vitro. PUSs have been linked to a wide range of diseases, including cancer. High expression of pre-mRNA Ψ synthases correlates with decreased survival in hepatocellular carcinoma cells (HCC) and rewiring of gene expression by alternative splicing is important for HCC pathology. NIH K99/R00 Pathway to Independence Award seeks to define PUS-dependent alternative splicing networks in HCC cells (Aim 1), identify molecular mechanisms by which Ψs regulates pre-mRNA splicing (Aim 2) and investigate mechanisms by which Ψ is deposited in pre-mRNA co-transcriptionally (Aim 3). During the mentored phase of this award Dr. Martinez will identify PUS-dependent alternative splicing in HepG2 cells by sequencing based methods for Ψ detection and alternative splicing analysis. splicing Dr. Martinez will determine the effect of Ψ on the binding of candidate Ψ-sensitive factors, whose binding sites are enriched for overlapping Ψ locations, using biochemical approaches. During the independent phase, Dr. Martinez will define the HCC specific pre-mRNA Ψ landscape and identify PUS-dependent HCC specific alternative splicing events by profiling Ψ, and corresponding splicing changes in primary normal and HCC cells. Her elucidation of mechanisms of Ψ-mediated splicing regulation in HCC cells will uncover potential disease related modes of splicing regulation. During the K99 and R00 portion of the award Dr. Martinez will determine how Ψ synthases are recruited to nascent pre-mRNA by investigating mechanisms of recruitment to chromatin and association with Pol II by a combination of chromatin- and co- immunoprecipitation experiments and measuring extent of pseudouridylation in relation to transcription dynamics using a novel sequencing approach. The pathology of disease-linked Ψ synthases may be due to dysregulation of their pre-mRNA targets. Therefore, defining and characterizing the effect of PUS-dependent pre-mRNA Ψs in pre-mRNA processing will help explain how these enzymes contribute to disease. The Pathway to Independence Award will allow Dr. Martinez to gain the necessary expertise (e.g. technology development and computational approaches to discover RNA modifications, techniques to study transcriptional coupling) and professional skills (e.g. mentoring and management) to become a competitive candidate for an academic position at a major research university.


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 = $0 )
2025	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	5	5/12/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $248,999 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	5	1/15/2024	NON-COMPETING CONTINUATION	$224,100
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	5	6/20/2024	NON-COMPETING CONTINUATION	$24,899
														Subtotal = $248,999

Issue Date FY: 2023 ( Subtotal = $248,999 )
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	4	2/14/2023	NON-COMPETING CONTINUATION	$24,899
2023	2023	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	4	1/13/2023	NON-COMPETING CONTINUATION	$224,100
														Subtotal = $248,999

Issue Date FY: 2022 ( Subtotal = $288,200 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	3	8/2/2022	EXTENSION WITH OR WITHOUT FUNDS	$39,200
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	3	1/31/2022	EXTENSION WITH OR WITHOUT FUNDS	$249,000
														Subtotal = $288,200

Grand Total All Awards = $786,198

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Role of pseudouridines in pre-mRNA processing

Award Number: R00GM135537

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: 08/01/2020

PERIOD OF PERFORMANCE END DATE: 01/31/2025

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