Intronic Sequences Influence Pre-mRNA Splicing of Acetylcholinesterase During Muscle Differentiation

Abstract

Acetylcholinesterase (AChE), an enzyme hydrolyzing the neurotransmitter acetylcholine, exists in multiple molecular forms originating from alternative splicing of a single gene. To study the influence of introns on AChE pre-mRNA splicing, we transiently transfected differentiating C2-C12 mouse myoblast cells with mouse AChE genomic constructs containing or devoid of each of the introns, and analyzed the mRNA species and gene products. Transfection of the genomic DNA resulted in overexpression of AChE mRNA and molecular forms normally seen in differentiated myotubes. However, such transfection in human embryonic kidney cells resulted in random splicing, indicating a tissue or species specific splicing selection. No exogenous AChE expression was evident when the first invariant intron was deleted, indicating the importance of the intron in gene transcription or mRNA stabilization. Transfection of a DNA construct devoid of invariant introns II and III, upstream of the alternative splice region, resulted in expression of alternatively spliced AChE mRNAs and corresponding molecular forms normally not seen in myotubes. Thus, these two introns either contain essential elements or confer a pre-mRNA structure that dictates downstream splicing preferences. Retention of either one of these introns resulted in a splicing pattern close to that of the genomic DNA indicating that presence of either invariantly spliced intron is sufficient for preventing random pre-mRNA splicing. To distinguish the influence of upstream splicing from that of a structural change on pre-mRNA splice selection, we deleted most of intron III in the intron II deleted construct, but kept the splice junctions and the branch point intact.