20] Principles and applications of a tat-based assay for analyzing specific RNA-protein interactions in mammalian cells

Abstract

Specific ribonucleic acid (RNA)–protein interactions are critical for the posttranscriptional regulation of cellular gene expression at the levels of messenger RNA (mRNA) splicing, nuclear export, translation, and stability and are key to the replication cycle of many pathogenic viruses, including human immunodeficiency virus type 1 (HIV-1), picornaviruses, and influenza viruses. Once a cis -acting RNA regulatory element has been defined, the next step is frequently the identification and subsequent characterization of the cellular or viral protein that determines the phenotype exerted by that element. In addition to the conceptually simple but often technically challenging approach of biochemical purification based on RNA affinity, an in vivo genetic approach can also be used to this problem. However, given that many of the interesting RNA–protein interactions occur in higher eukaryotes and, more specifically, in human cells, it is apparent that a simple and sensitive assay for RNA–protein binding in these same cells would be of considerable utility. The Tat-based assay for analyzing specific RNA–protein interactions relies on the unusual ability of the HIV-1 Tat protein to potently activate transcription from the viral long terminal repeat (LTR) promoter element when targeted to a promoter-proximal RNA target sequence. Specific RNA binding by a Tat-protein Y chimera can in principle be readily detected by cotransfection of an appropriate indicator construct and chimeric Tat protein expression plasmid into a relevant cell line.