Interactions of INS (CRS) elements and the splicing machinery regulate the production of Rev-responsive mRNAs

The human immunodeficiency virus type (HIV-1) Rev protein stimulates the export to the cytoplasm of unspliced HIV-1 mRNAs carrying the Rev response element (RRE). However, simple addition of the RRE to beta-globin pre-mRNA does not confer a Rev response on this heterologous transcript. In this paper, we demonstrate that a strong Rev response is conferred on beta-globin pre-mRNA when an inhibitory (INS) element is inserted into the gene together with the RRE. In the presence of INS element, Rev was able to stimulate the export to the cytoplasm of unspliced mRNA 10 to 15-fold. INS elements from the HIV-1 p17 gag and pol genes were equally active in complementing Rev-dependent nuclear export of unspliced mRNA. By contrast, mutated p17 gag INS element, known to be inactive in gag mRNA instability assays, was unable to complement the Rev/RRE system and stimulate nuclear export. Similarly, AUUUA-instability elements from the granulocyte-macrophage colony stimulating factor mRNA (GM-CSF) destabilised beta-globin mRNA but could not substitute for the HIV INS elements. Complementation between the Rev/RRE system and the INS elements was only observed when splicing was efficient. When splicing of the beta-globin gene receptor is impaired by mutations in the 5' splice donor, the 3' splice acceptor sequence, or the polypyrimidine tract, the majority of the unspliced mRNA is exported from the nucleus in the absence of Rev. In the presence of splice site mutations, Rev is able to act independently of a functional INS element and increase the export of unspliced mRNA three to fivefold. We propose that nuclear factor(s) binding to INS elements separate unspliced beta-globin pre-mRNA from the splicing apparatus. Pre-mRNA in this "INS compartment" remains accessible to Rev. Thus, there is a synergy between the INS elements and Rev which leads to enhanced nuclear export of unspliced mRNA.