Kevin Czaplinski - US grants

The proposed research project aims to identify the putative additional factors required for the export of U snRNAs from the nucleus. Understanding the processes of nucleocytoplasmic transport are of central importance to the control of gene expression in eukaryotes, and understanding this process may provide new targets for treatment of disease. Viral infections which require entry of the virion into the nucleus, or export of viral products from the nucleus could be envisioned to provide a means to block production of crucial viral components, thus preventing harmful viral infections. The current data regarding U snRNA export suggests the presence of at least one additional factor for U snRNA export which may be regulated at the level of nucleocytoplasmic transport. By a combination of in vivo microinjection of Xenopus oocytes, and in vitro biochemistry, the experiments designed in this research proposal will uncover additional factors required for reconstitution of U snRNA export from Xenopus oocytes which have been compromised in this cellular process. Once identified the role this factor plays in the export of U snRNA and other RNAs will be characterized in Xenopus and other organisms.

Cellular signals in the nervous system create a large demand for new proteins at synapses far from the cell body. Neurons interpret these signals and increase protein synthesis (translation) near the synapses in axons and dendrites. This translation localized to axons and dendrites supports proper nervous system development and repair as well as basic brain functions including learning, memory. Importantly, the specific proteins that are synthesized locally in dendrites and axons is specified by mRNA sequences that specify trafficking to these distinct locations. Despite this key relationship of mRNA trafficking to brain function, many fundamental principles of how mRNA trafficking occurs and is regulated are unknown. Studies of mRNA trafficking within the cytoplasm pose unique challenges and require single cell, even single molecule analysis in real time in living cells. This project develops an accessible single molecule assay with a well-documented mRNA as a paradigm that can be applied to training of graduate, undergraduate and high school students. Using these assays the investigators will correlate changes in mRNA movement that occur after interference with cis-acting sequences or trans-acting factors that affect the trafficking of this mRNA in growing neurons. These results will uncover the basis for how mRNA traffics to sites of translation.

The integration of the research with the training of the next generation of scientists is important to the broader impact of this proposal. This project will fundamentally advance our understanding of neuronal cell biology while at the same time train the highest quality scientist/educators for this area of research that is so important to the future scientific efforts of the country.