INTERACTIONS OF INS (CRS) ELEMENTS AND THE SPLICING MACHINERY REGULATE THE PRODUCTION OF REV-RESPONSIVE MESSENGER-RNAS

The human immunodeficiency virus type 1 (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-g lobin pre-mRNA does not confer a Rev response on this heterologous tra nscript. In this paper, we demonstrate that a strong Rev response is c onferred on beta-globin pre-mRNA when an inhibitory (INS) element is i nserted into the gene together with the RRE. In the presence of the IN S element, Rev was able to stimulate the export to the cytoplasm of un spliced mRNA 10 to 15-fold. INS elements from the HIV-1 p17 gag and po l genes were equally active in complementing Rev-dependent nuclear exp ort of unspliced mRNA. By contrast, mutated p17 gag INS element, known to be inactive in gag mRNA instability assays, was unable to compleme nt the Rev/RRE system and stimulate nuclear export. Similarly, AUUUA-i nstability elements from the granulocyte-macrophage colony stimulating factor mRNA (GM-CSF) destabilised beta-globin mRNA but could not subs titute for the HIV INS elements. Complementation between the Rev/RRE s ystem and the INS elements was only observed when splicing was efficie nt. When splicing of the beta-globin gene receptor is impaired by muta tions in the 5' splice donor, the 3' splice acceptor sequence, or the polypyrimidine tract, the majority of the unspliced mRNA is exported f rom the nucleus in the absence of Rev. In the presence of splice site mutations, Rev is able to act independently of a functional INS elemen t and increase the export of unspliced mRNA three to fivefold. We prop ose that nuclear factor(s) binding to INS elements separate unspliced beta-globin pre-mRNA from the splicing apparatus. Pre-mRNA in this ''I NS compartment'' remains accessible to Rev. Thus, there is a synergy b etween the INS elements and Rev which leads to enhanced nuclear export of unspliced mRNA. (C) 1996 Academic Press Limited