Gersappe A, Pintel D J
Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia, Columbia, Missouri 65212, USA.
Mol Cell Biol. 1999 Jan;19(1):364-75. doi: 10.1128/MCB.19.1.364.
The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5'-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5' and 3' regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3' splice site upstream (i.e., the exon 5' terminus) or a strong 5' splice site downstream (i.e., the exon 3' terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3' splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3' splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5' splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.
小鼠细小病毒（MVM）的290个核苷酸的NS2特异性外显子通过可变剪接产生，其上游有一个大内含子，下游有一个小内含子，它在R1 mRNA中作为一个大的5'末端外显子的一部分组成性出现（在开放阅读框3 [ORF3]中进行翻译），并在R2 mRNA中作为一个内部外显子出现（在ORF2中进行翻译）。我们鉴定出一种新型的双组分外显子增强子元件，它分别由外显子5'和3'区域内富含CA和富含嘌呤的元件组成，在体内成熟的剪接mRNA中包含NS2特异性外显子序列时该元件是必需的。这两个组成不同的增强子元件在功能上有一定冗余：单独一个元件至少能部分支持外显子的包含。它们也是可互换的：任何一个元件在任何一个位置都能发挥作用。上游（即外显子5'末端）的一个强3'剪接位点或下游（即外显子3'末端）的一个强5'剪接位点足以防止NS2特异性外显子的跳跃，并且将NS2特异性外显子作为内部外显子包含到成熟的、经过两次剪接的R2 mRNA中需要一个功能性的上游3'剪接位点。双组分增强子在功能上强化了这些末端：上游内含子3'剪接位点的多嘧啶序列的改善可以克服对富含CA和富含嘌呤元件两者的需求，并且富含嘌呤的元件也支持通过下游5'剪接位点介导的外显子包含。总之，一个次优的大内含子多嘧啶序列、下游小内含子内的序列以及一个新型的双组分外显子增强子共同作用，以产生在体内观察到的R1和R2的平衡水平。我们认为，这种不寻常的双组分外显子增强子的功能是介导NS2特异性外显子在单次剪接的R1和两次剪接的R2中都能有适当水平的包含。
