An assay to measure the activity of RNA elements and proteins that promote the expression of mRNA with retained introns

Abstract

The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability ofcis-andtrans-acting factors to promote the export and translation of mRNA with retained introns.One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.