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 o f at least one intron. We previously showed that addition of almost an y of the introns from the mouse thymidylate synthase (TS) gene to an i ntronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The go al 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 signifi cant increase in expression, suggesting that its inability to stimulat e expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removin g purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within th e intron each had little effect on the level of expression. However, w hen 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 g-fold. When the splice donor site of TS intr on 1 (a stimulatory intron) was changed to that of TS intron 4, the mo dified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that int rons can stimulate gene expression by a process that depends directly on the splicing reaction.