Inclusion of the NS2-specific exon in minute virus of mice mRNA is facilitated by an intronic splicing enhancer that affects definition of the downstream small intron

Alternative splicing of pre-mRNAs plays a critical role in maximizing the c oding capacity of the small parvovirus genome. The small-intron region of m inute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlap ping alternative splicing, using two donors, D1 and D2, and two accepters, Al and A2, within a region of 120 nucleotides, that governs the steady-stat e ratios of the various viral mRNAs. In a previous report we demonstrated t hat a complex interaction between both donor and acceptor sequences, as wel l as the constraints of size, defines the small intron and governs its alte rnative splicing. We also identified a G-rich intronic splicing enhancer se quence (IES) that appeared to function as both an intron- and an exon-defin ing element. In this report we further examined the components that govern MVM small-intron splicing. In fully processed wild-type mRNAs, Al is used p referentially over A2. In this report, we show that in the context of the w ild-type small intron the position of the downstream acceptor A2 was prefer red, and the primary sequence of Al must be stronger for it to be utilized at wild-type efficiency. Use of A2 in generation of the minor spliced forms D2/A2 required the IES because of a weak A2 polypyrimidine tract and becau se of the relative strength of Al. The small size of the intron and the rel ative position of the IES were also shown to play a critical role in donor and acceptor site selection. Finally, we have further characterized how the IES functions as an intronic enhancer of upstream exon definition. When th e small intron was expanded, upstream exon inclusion was dependent upon the position of the IES. Within the context of the small intron, alterations o f the small intron that overcame the requirement for the IES for splicing t o A2 also permitted wild-type levels of upstream exon inclusion in the abse nce of the IES, suggesting that, in its natural context, the IES facilitate s upstream exon inclusion by affecting small-intron definition (C) 1999 Aca demic Press.