SPECIFICITY OF RNA TRANSPORT IN VITRO

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Mechanisms controlling gene expression in eukaryotic cells are complex, encompassing transcriptional and post-transcriptional components. Due to inherent difficulties in their study, post-transcriptional mechanims have received relatively less attention; in particular, RNA transport has been studied as the appearance of rapidly-labeled RNA in the supernate after incubation of nuclei in vitro. Such assays would be more firmly accepted if the transported RNA was better characterized. It is the intent of this proposal to test the fidelity of carefully selected in vitro system by examining the behavior of specific sequences. Using perturbations which result in great differences in mRNA formation and abundance in vivo, metabolism of an acute phase reactant sequence (alpha1-acid glycoprotein), a hormonally-responsible globulin sequence (alpha2Mu-globulin), and marker sequences for mature (albumin) and primitive (Alpha-fetoprotein) liver cells will be examined with an in vitro transport system. An additional model will employ an analbuminemic rat strain, which transcribes albumin sequences into nuclear RNA but restricts them to the nucleus. Transported and nucleus-restricted RNA will be isolated and in vitro translation products will be examined. RNA will be separated by size electrophoretically, "Northern blotted" and hybridized with specific P32-cDNA probes, and autoradiographs will be developed. Mixing experiments will document and quantitate cytoplasmic contamination of nuclear and transported preparation. These experiments will determine whether in vitro transport assays can support processing/transport of correctly-sized functional mRNA, with modulation paralleling in vivo circumstances. Providing specificity can be established (as preliminary results indicate), use of in vitro transport assays should contribute valuably to understanding controls of gene expression, especially the altered post-transcriptional RNA transport associated with carcinogenesis. If specificity cannot be established, the considerable effort and funding devoted to their use in studies of "in vivo specificity" should be curtailed.