THE EXON SPLICING SILENCER IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TATEXON-3 IS BIPARTITE AND ACTS EARLY IN SPLICEOSOME ASSEMBLY

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RN A is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals with in the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silence rs (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit spli cing at the 3' splice site flanking tat exon 2. This occurs at an earl y step in spliceosome assembly. Splicing at the 3' splice site flankin g tat exon 3 is regulated by a bipartite element composed of an ESE an d an ESS (ESS3). Here we show that ESS3 is composed of two smaller ele ments (AGAUCC and UUAG) that can inhibit splicing independently. We al so show that ESS3 is more active in the context of a heterologous subo ptimal splice site than of an optimal 3' splice site. ESS3 inhibits sp licing by blocking the formation of a functional spliceosome at an ear ly step, since A complexes are not detected in the presence of ESS3. C ompetitor RNAs containing either ESS2 or ESS3 relieve inhibition of sp licing of substrates containing ESS3 or ESS2. This suggests that a com mon cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.