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 coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing, using two donors, D1 and D2, and two acceptors, A1 and A2, within a region of 120 nucleotides, that governs the steady-state ratios of the various viral mRNAs. In a previous report we demonstrated that a complex interaction between both donor and acceptor sequences, as well as the constraints of size, defines the small intron and governs its alternative splicing. We also identified a G-rich intronic splicing enhancer sequence (IES) that appeared to function as both an intron- and an exon-defining element. In this report we further examined the components that govern MVM small-intron splicing. In fully processed wild-type mRNAs, A1 is used preferentially over A2. In this report, we show that in the context of the wild-type small intron the position of the downstream acceptor A2 was preferred, and the primary sequence of A1 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 because of the relative strength of A1. The small size of the intron and the relative 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 the small intron was expanded, upstream exon inclusion was dependent upon the position of the IES. Within the context of the small intron, alterations of the small intron that overcame the requirement for the IES for splicing to A2 also permitted wild-type levels of upstream exon inclusion in the absence of the IES, suggesting that, in its natural context, the IES facilitates upstream exon inclusion by affecting small-intron definition.
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral; D004742:Enhancer Elements, Genetic; D005091:Exons; D007438:Introns; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D011743:Pyrimidines; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins / Q000235:genetics*