PRESENCE OF EXON SPLICING SILENCERS WITHIN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT EXON-2 AND TAT-REV EXON-3 - EVIDENCE FOR INHIBITION MEDIATED BY CELLULAR FACTORS

Human immunodeficiency virus type 1 (HIV-1)pre-mRNA splicing is regula ted in order to maintain pools of unspliced and partially spliced vira l RNAs as well as the appropriate levels of multiply spliced mRNAs dur ing virus infection. We have previously described an element in fat ex on 2 that negatively regulates splicing at the upstream fat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, M ol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicin g enhancer (ESE) elements, we have termed this element an exon splicin g silencer (ESS). We show evidence for another negative cis-acting reg ion within fat-rev exon 3 of HIV-1 RNA that has sequence motifs in com mon with a 20-nt ESS element in tat exon 2. This sequence is juxtapose d to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream fat-rev 3' splice site. Inhibition of the spl icing of substrates containing the ESS element in fat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing me diated by the ESS can be specifically abrogated by the addition of com petitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and fat-rev exon 3.