A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF(65) and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

牛乳头瘤病毒1型（BPV-1）的外显子剪接抑制子（ESS）紧接在BPV-1剪接增强子1的下游，可负调控位于核苷酸3225处的次优3'剪接位点的选择。本研究证实，这种富含嘧啶的ESS通过阻断剪接体组装的早期步骤，抑制上游3'剪接位点的使用。对结合ESS的蛋白质进行的分析显示：富含尿嘧啶的5'区域可结合U2AF(65)与多嘧啶束结合蛋白；富含胞嘧啶的中央区域可结合35kDa及54~55kDa的丝氨酸/精氨酸富集（SR）蛋白；而富含腺嘌呤/鸟嘌呤的3'末端则可结合可变剪接因子/剪接因子2。突变与功能研究表明，ESS中最关键的区域定位于富含胞嘧啶的核心序列（GGCUCCCCC）。该核心序列与额外的下游非特异性核苷酸共同存在时，即可部分抑制BPV-1前体mRNA的剪接体组装与剪接过程。当过量添加纯化的HeLa细胞SR蛋白时，ESS介导的剪接抑制可被部分逆转，这表明ESS通过干扰SR蛋白的正常桥接与招募活性，从而抑制前体mRNA的剪接。