Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.     

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and 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 between 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 the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.