SPLICING OF 5' INTRONS DICTATES ALTERNATIVE SPLICE SELECTION OF ACETYLCHOLINESTERASE PRE-MESSENGER-RNA AND SPECIFIC EXPRESSION DURING MYOGENESIS

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acety lcholinesterase (AChE) pre-mRNA results in expression of the prevailin g enzyme species in the nervous system and at the neuromuscular juncti on of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selec tive intron deletion from and site-directed mutagenesis in the Ache ge ne. Transfection of a plasmid lacking two invariant introns (introns I I and III) within the open reading: frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced m RNAs encoding enzyme forms not found endogenously in myotubes, Retenti on of either intron II or III is sufficient to control the tissue-spec ific pre-mRNA splicing pattern prevalent in situ. Further deletions an d branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron bet ween the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which t he alternative intron influences the fidelity of correct upstream spli cing, which, in turn, determines the downstream splice selection of al ternative exons.