CA- and purine-rich elements form a novel bipartite exon enhancer which governs inclusion of the minute virus of mice NS2-specific exon in both singly and doubly spliced mRNAs

The alternatively spliced 290-nucleotide NS2-specific exon of the parvoviru s minute virus of mice (MVM), which is banked by a large intron upstream an d 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 fr ame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translat ed in ORF2). We have identified a novel bipartite exon enhancer element, co mposed 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 seque nces in mature spliced mRNA in vivo. These two compositionally different en hancer elements are somewhat redundant in function: either element alone ca n 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 downs tream (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 fo r inclusion of the NS2-specific exon as an internal exon into the mature, d oubly spliced R2 mRNA. The bipartite enhancer functionally strengthens thes e termini: the requirement for both the CA rich and purine-rich elements ca n be overcome by improvements to the polypyrimidine tract of the upstream i ntron 3' splice site, and the purine-rich element also supports exon inclus ion mediated through the downstream 5' splice sites. In summary, a suboptim al large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield th e balanced levels of R1 and R2 observed in vivo. We suggest that the unusua l bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.