Si Z H, Rauch D, Stoltzfus C M
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Mol Cell Biol. 1998 Sep;18(9):5404-13. doi: 10.1128/MCB.18.9.5404.
Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.
人类免疫缺陷病毒1型（HIV-1）RNA的低效剪接对于保留未剪接和单剪接的病毒RNA以通过Rev依赖途径转运至细胞质是必要的。HIV-1基因组中控制剪接速率的信号包括弱3'剪接位点、外显子剪接增强子（ESE）和外显子剪接沉默子（ESS）。我们先前已经表明，tat外显子2内存在的一个ESS（ESS2）和一个次优3'剪接位点共同作用，抑制tat外显子2侧翼3'剪接位点的剪接。这发生在剪接体组装的早期步骤。tat外显子3侧翼3'剪接位点的剪接由一个由ESE和一个ESS（ESS3）组成的二分元件调节。在这里我们表明，ESS3由两个较小的元件（AGAUCC和UUAG）组成，它们可以独立抑制剪接。我们还表明，在异源次优剪接位点的背景下，ESS3比在最佳3'剪接位点的背景下更具活性。ESS3通过在早期步骤阻断功能性剪接体的形成来抑制剪接，因为在存在ESS3的情况下未检测到A复合物。含有ESS2或ESS3的竞争RNA可缓解对含有ESS3或ESS2的底物剪接的抑制。这表明ESS2和ESS3介导的tat mRNA剪接抑制可能需要一种共同的细胞因子。
