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Interrogating spatiotemporal control of mRNA in Drosophila morphogenesis using Cas13-based techniques

Award Number: F30NS137764
ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 07/01/2024
PERIOD OF PERFORMANCE END DATE: 06/30/2029

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Interrogating spatiotemporal control of mRNA in Drosophila morphogenesis using Cas13-based techniques - PROJECT SUMMARY/ABSTRACT Establishing subcellular compartments and cell polarity requires asymmetric distribution of the proteome. mRNA localization and onsite translation ensure that cells produce proteins at the right place and time, and this regulation allows for asymmetric distribution of proteins for morphogenesis. In particular, the development, maintenance, and function of neurons require posttranscriptional regulation as the complex and polarized morphology necessitates the transport of mRNAs to neurites, which can be as long as a meter in animals. Mutations in RNA binding proteins that regulate mRNA localization and onsite translation are associated with neurological diseases, including amyotrophic lateral sclerosis, spinal motor atrophy, and fragile X syndrome. In these diseases, mutations in RNA binding proteins are also associated with abnormal neuron morphology. Despite the knowledge that neuronal morphogenesis requires proper mRNA localization, the mechanistic link between spatiotemporal regulation of mRNA and morphogenesis remains unknown. This proposal investigates the mechanistic link between spatiotemporal regulation of mRNA and neuronal remodeling during morphogenesis. I will take advantage of Drosophila mushroom body and dendritic arborization neurons as models of developmental neuronal remodeling, where early neuronal connections degenerate and neurites are refined. Specifically, I will investigate spatial and temporal regulation of the chickadee (chic) transcript during regrowth of dendrites in Drosophila class IV dendritic arborization neurons and mushroom body γ neurons. I hypothesize that during the neurite extension phase of neuronal remodeling of these PNS and CNS neurons, respectively, chic mRNA localizes to branch emergence sites where its cohort of protein interactors changes to anchor the transcript and to alter translation rates. First, I will characterize chic localization and motility during neuronal remodeling using a novel Cas13-based method for live imaging. Second, I will identify proteins interacting with chic mRNA during neuronal remodeling using a Cas13-based method for RNA-centric proximity labeling. CRISPR/Cas13-based methods are an exciting, novel approach to probe endogenous RNA in live organisms and unlike established methods of probing RNA, they do not perturb the transcript sequences. CRISPR methods are generally very precise, and the family of Cas13 proteins I will use is highly specific. The results of this proposal will elucidate the role of spatiotemporal regulation of mRNA in the regulation of gene expression needed for neuronal outgrowth. Additionally, I will establish Cas13-based methods that can be used for investigating RNA for other biological questions and in other organisms.


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: 2026 ( Subtotal = $55,114 )
2026	2026	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	3	7/27/2026	NON-COMPETING CONTINUATION	$55,114
														Subtotal = $55,114

Issue Date FY: 2025 ( Subtotal = $54,538 )
2025	2025	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	2	6/5/2025	NON-COMPETING CONTINUATION	$54,538
														Subtotal = $54,538

Issue Date FY: 2024 ( Subtotal = $53,974 )
2024	2024	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	001	1	6/10/2024	NEW	$0
2024	2024	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	002	1	8/6/2024	NEW	$0
2024	2024	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	6/10/2024	NEW	$53,974
														Subtotal = $53,974

Grand Total All Awards = $163,626

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Interrogating spatiotemporal control of mRNA in Drosophila morphogenesis using Cas13-based techniques

Award Number: F30NS137764

ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 07/01/2024

PERIOD OF PERFORMANCE END DATE: 06/30/2029

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