The ultimate goal of this project is to understand the molecular mechanisms by which RNA processing is controlled by signaling pathways, and the impact of this regulation on cellular function. The specific focus of the current funding period is on regulation of alternative pre-mRNA splicing during T cell activation. Alternative splicing is an essential and ubiquitous mechanism of gene regulation, which allows for the diversification and control of protein expression in distinct cell-types or environmental conditions. In particular, itis well established that signal-induced alternative splicing is pervasive during neuronal activity, nutrient sensing, oncogenesis and immune function. However, a mechanistic view of how signaling pathways control alternative splicing remains lacking. T cell activation provides an excellent model system for complex cellular responses, in that multiple signaling pathways are triggered downstream of antigen engagement and act, individually and cooperatively, to induce T cell effector functions. Importantly, several hundred genes are known to undergo alternative splicing in response to T cell activation; however, it remains to be determined how these genes are regulated, which genes are co-regulated by overlapping mechanisms/pathways, and what the ultimate functional consequence is of such regulation. This proposal will address these unanswered questions of signal- induced alternative splicing by leveraging recently developed methodologies and systems to determine: (1) the specific signaling pathways that lead to changes in alternative splicing following antigen stimulation of T cells, and the RNA binding proteins (RBPs) that connect each signaling pathway with their respective splicing targets; (2) the molecular mechanisms by which RBPs and/or cell signaling regulate specific transitions in spliceosome assembly to direct isoform expression; (3) the functional impact of alternative splicing on cell signaling through regulating isoform expression of related kinases; and (4) how regulation of alternative splicing is coordinated with other RNA biogenesis events such as transcription and 3' end processing. Together these studies will provide novel insight regarding the interplay of signaling and splicing in shaping cellular function during T cell activation. Sinc the signaling pathways induced upon T cell activation are also functional in pathways related to cell growth, metabolism and cancer, the insight gained in these studies will be broadly applicable to numerous cellular responses far beyond the immune system. In addition, the studies proposed here will reveal new paradigms regarding the molecular mechanisms by which cells regulate spliceosome assembly, and how RBPs coordinately control splicing along with other steps in RNA processing. Results from these experiments will thus significantly increase understanding of the mechanisms that control alternative splicing, an essential and ubiquitous process for regulating gene expression across all human cell types.

Public Health Relevance

To function appropriately and prevent disease states, all cells in the human body must be able to regulate what proteins they express in response to environmental cues or need. A pervasive mechanism for regulating protein expression and cellular function in response to extracellular triggers is that of signal- induced alternative splicing. The studies in this proposal seek to elucidate the molecular mechanisms by which changes in environment regulate alternative splicing and to determine the consequences of such regulation for maintaining proper cellular function and human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
5R35GM118048-02
Application #
9271211
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Bender, Michael T
Project Start
2016-05-09
Project End
2021-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Black, Kathryn L; Naqvi, Ammar S; Asnani, Mukta et al. (2018) Aberrant splicing in B-cell acute lymphoblastic leukemia. Nucleic Acids Res 46:11357-11369
Thompson, Matthew G; Muñoz-Moreno, Raquel; Bhat, Prasanna et al. (2018) Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing. Nat Commun 9:2407
Agosto, Laura M; Lynch, Kristen W (2018) Alternative pre-mRNA splicing switch controls hESC pluripotency and differentiation. Genes Dev 32:1103-1104
Shinde, Mansi Y; Sidoli, Simone; Kulej, Katarzyna et al. (2017) Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing. J Biol Chem 292:18240-18255
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Ajith, Sandya; Gazzara, Matthew R; Cole, Brian S et al. (2016) Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells. RNA Biol 13:569-81
Vaquero-Garcia, Jorge; Barrera, Alejandro; Gazzara, Matthew R et al. (2016) A new view of transcriptome complexity and regulation through the lens of local splicing variations. Elife 5:e11752