STIMULATION OF GENE-EXPRESSION BY INTRONS - CONVERSION OF AN INHIBITORY INTRON TO A STIMULATORY INTRON BY ALTERATION OF THE SPLICE DONOR SEQUENCE
STIMULATION OF GENE-EXPRESSION BY INTRONS - CONVERSION OF AN INHIBITORY INTRON TO A STIMULATORY INTRON BY ALTERATION OF THE SPLICE DONOR SEQUENCE
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DOI：
10.1093/nar/21.25.5901
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发表时间：
1993-12-25
影响因子：
14.9
通讯作者：
JOHNSON, LF
中科院分区：
文献类型：
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作者：
KORB, M;KE, YB;JOHNSON, LF
许多哺乳动物基因的有效表达依赖于至少一个内含子的存在。我们以前表明，除了几乎任何内含子从小鼠胸苷酸合成酶（TS）基因的一个无内含子的TS小基因导致了大量的表达增加。然而，增加内含子4导致小基因表达减少。本研究的目的是确定为什么TS内含子4不能刺激表达。内含子4插入到核糖体蛋白L32基因的内含子依赖性衍生物中并没有导致表达的显著增加，这表明其不能刺激表达是由于内含子内的序列。删除内含子4的大部分内部，改善推定的分支点，从内含子3'端的嘧啶段去除嘌呤，或去除内含子内可能的可变剪接受体或供体位点，每一个对表达水平几乎没有影响。然而，当内含子4的剪接供体序列被修改，使其与U1 snRNA完全互补时，修改后的内含子4刺激表达约g倍。当TS内含子1（刺激性内含子）的剪接供体位点改变为TS内含子4的剪接供体位点时，修饰的内含子1非常低效地剪接，并且失去刺激mRNA产生的能力。我们的观察结果支持这样的观点，即内含子可以通过直接依赖于剪接反应的过程来刺激基因表达。
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 g-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.