The broad, long-term goals of this project are to characterize how platelets regulate prolonged inflammatory events that dictate the course of human disease. An immediate focus is to define how platelets regulate functional responses via signal-dependent translation, a previously uncharacterized trait of platelets. In the last funding period we identified pathways for signal-dependent translation of critical mRNAs in platelets that were previously unrecognized and have physiologic significance. The current application builds on these discoveries and uses human megakaryocytes, proplatelets, and platelets to characterize a novel upstream checkpoint in the translational control pathway referred to as signal-dependent splicing. The central hypothesis is that megakaryocytes deliver their nuclear-based spliceosome to proplatelets during thrombopoiesis, a complex used by platelets to process precursor mRNAs into viable, translatable mRNAs. The first specific aim characterizes the spliceosome in megakaryocytes and proplatelets. In doing so, aim one tracks critical spliceosome factors during thrombopoiesis, determines if integrins regulate spliceosomal transfer, and resolves whether spliceosomal components exit the nucleus via regulated export programs or passive diffusion through a damaged nuclear envelope.
The second aim characterizes the role of the spliceosome in mature, human platelets.
This aim defines requisite spliceosomal factors and delineates whether integrins control signal-dependent splicing events in platelets.
Aim two finishes by identifying pre-mRNAs in platelets that are spliced in response to external cues. Importantly, aim two will test the hypothesis that newly spliced mRNAs comprise a subgroup of transcripts in activated platelets that code for critical inflammatory proteins. Together, the proposed studies focus on a new biological function of platelets, challenge existing paradigms of thrombopoiesis, and identify a novel mode of nuclear-independent post-transcriptional gene regulation that may have a correlative role in other cells of the vascular system. ? ?
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