Thursday, November 21, 2024 11/21/2024

Understanding the mechanisms that regulate cytoplasmic capping and defining its contributions to post-transcriptional gene regulation

Award Number: R35GM137819
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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R35_Project Summary/Abstract The N7-methylguanosine (m7G) cap is a unique molecular identifier that is a focal point for post-transcriptional gene regulatory pathways. The m7G cap serves as both a roadblock to enzymes that would degrade the mRNA and a landing pad for cap binding proteins that coordinate the pre-mRNA processing, nuclear export, and translation initiation of most mRNAs. Until recently, capping was thought to be exclusively nuclear, and decapping was thought to irreversibly destine the RNA to degradation. Simply stated, cytoplasmic capping is the process by which an m7G cap is returned to an uncapped mRNA in the cytoplasm. Cytoplasmic capping requires NCK1 to coordinate the sequential actions of an unknown kinase, the capping enzyme, and an RNA methyltransferase, which phosphorylate and cap the targeted mRNA and methylate the newly-added cap respectively. Although we have learned much about the biochemistry of cytoplasmic capping, many fundamental questions remain unanswered. The hypotheses driving this proposal are that: (1) Specific RNA sequence elements (or modifications) recruit and/or trigger cytoplasmic capping activity and that (2) the cytoplasmic capping of 5’-truncated mRNAs serves as a new tier of post-transcriptional gene regulation. This study will seek answers to three key questions. First, a combination of data mining and new sequencing experiments will uncover the sequences that target an mRNA to the cytoplasmic capping machinery. A bioinformatics approach integrating published data sets marking cap positions and transcription start sites (TSS) will identify non-TSS- associated caps. Oxford Nanopore direct RNA sequencing will then compare RNA harvested from cells +/- dominant negative cytoplasmic capping components to map full-length mRNA sequences. The synthesis of these studies should ascertain the 5’ ends, the alternative splicing patterns, and polyadenylation site choices of cytoplasmically capped mRNAs. Second, CRISPR knockouts of mRNA decapping enzymes (Dcp2, DcpS, etc) and candidate endonucleases will identify the cellular mechanism(s) that generate uncapped ends for the cytoplasmic capping machinery. These knockouts will be paired with focused and transcriptome-wide methods to validate changes in cytoplasmic capping. Third, a combination of in vivo RNA labeling experiments and ribosome profiling will establish how cytoplasmic capping surveys mRNAs during the onset of the stress response. The generation, cytoplasmic capping, and translation of 5’-truncated mRNAs into N-terminally- shortened proteins would effectively be a new tier of post-transcriptional gene regulation with far-reaching impacts on the function(s) of the N-terminally truncated proteins. To summarize, this work will (1) identify and validate the sequences that regulate cytoplasmic capping (2) determine the mechanism(s) by which RNA substrates are generated for cytoplasmic capping, and (3) understand the in vivo function(s) of cytoplasmic capping during the onset of acute stress responses.


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: 2024 ( Subtotal = $302,960 )
2024	2024	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	000	5	6/12/2024	NON-COMPETING CONTINUATION	$403,750
2024	2023	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	001	4	6/27/2024	SUPPLEMENT FOR EXPANSION	-$100,790
														Subtotal = $302,960

Issue Date FY: 2023 ( Subtotal = $517,436 )
2023	2023	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	000	4	6/15/2023	NON-COMPETING CONTINUATION	$403,750
2023	2023	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	001	4	6/16/2023	SUPPLEMENT FOR EXPANSION	$113,686
														Subtotal = $517,436

Issue Date FY: 2022 ( Subtotal = $585,825 )
2022	2022	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	004	3	7/6/2022	SUPPLEMENT FOR EXPANSION	$124,021
2022	2022	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	002	3	6/30/2022	SUPPLEMENT FOR EXPANSION	$23,860
2022	2022	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	003	3	7/1/2022	SUPPLEMENT FOR EXPANSION	$23,860
2022	2022	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	001	3	6/29/2022	NON-COMPETING CONTINUATION	$403,750
2022	2022	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	000	2	5/24/2022	SUPPLEMENT FOR EXPANSION	$10,334
														Subtotal = $585,825

Issue Date FY: 2021 ( Subtotal = $451,472 )
2021	2021	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	001	2	6/24/2021	SUPPLEMENT FOR EXPANSION	$23,860
2021	2021	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	002	2	6/25/2021	SUPPLEMENT FOR EXPANSION	$23,862
2021	2021	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	000	2	6/18/2021	NON-COMPETING CONTINUATION	$403,750
														Subtotal = $451,472

Issue Date FY: 2020 ( Subtotal = $392,373 )
2020	2020	METHODIST HOSPITAL, THE	6670 BERTNER AVE	HOUSTON	TX	77030	HARRIS	USA	Biomedical Research and Research Training	000	1	8/3/2020	NEW	$392,373
														Subtotal = $392,373

Grand Total All Awards = $2,250,066

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Understanding the mechanisms that regulate cytoplasmic capping and defining its contributions to post-transcriptional gene regulation

Award Number: R35GM137819

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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