Identification of IN Vivo Mrna Ligands of Hnrnp A2

Brooks, Seth

Abstract

The study of trans-acting factors important in post-translational gene regulation has been limited by the difficulty of identifying these mRNA ligands in vivo. We have previously identified hnRNP A2 as specifically binding GLUT-1 and VEGF mRNA in vivo, and recent data indicates that it also binds to thioredoxin reductase mRNA in vivo. All of the genes as well as hnRNP A2 on thioredoxin reductase will provide insight into their roles in malignant transformation. One a more general level, identification of the mRNA ligand of hnRNP A2 will improve the understanding of mRNA-hnRNP interactions which in turn could help to resolve the difference in binding affinities of different hnRNPs to similar cis-acting mRNA elements. To identify mRNAs specifically bound by hnRNP A2, polysomes from 293 cells over-expression FLAG tagged hnRNP A2, hnRNP A1, or cytoplasmically localizing mutants, are immunprecipitated and used as probe to screen a 293 cell line cDNA library. DNA sequencing is used to identify positive clones and unknown clones are fully sequenced. Further analysis will examine the turnover rates of thioredoxin reductase, as well as other identified messages, in the presence of over-expressed hnRNP A2, confluence, cell growth and hypoxia. The results of this messages, in the presence of over-expressed hnRNP A2, confluence, cell growth and hypoxia. The results of this study will help elucidate the impact of hnRNP A2 on the half-life of thioredoxin reductase and could provide insight into the role of hnRNP A2 as well as thioredoxin in various neoplasms, as well as potentially provide a site for chemotherapeutic treatment, either direct at hnRNP A2 or downstream at the protein products of the mRNA it binds.