ORDER OF INTRON REMOVAL DURING SPLICING OF ENDOGENOUS ADENINE PHOSPHORIBOSYLTRANSFERASE AND DIHYDROFOLATE-REDUCTASE PREMESSENGER RNA
ORDER OF INTRON REMOVAL DURING SPLICING OF ENDOGENOUS ADENINE PHOSPHORIBOSYLTRANSFERASE AND DIHYDROFOLATE-REDUCTASE PREMESSENGER RNA
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DOI：
10.1128/mcb.13.10.6211
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发表时间：
1993-10-01
影响因子：
5.3
通讯作者：
CHASIN, LA
中科院分区：
文献类型：
--
作者：
KESSLER, O;JIANG, YJ;CHASIN, LA
利用逆转录和聚合酶链反应，我们确定了中国仓鼠卵巢细胞内源性腺嘌呤磷酸核糖基转移酶（aprt）基因的4个内含子的剪接顺序。该方法包括对保留一个内含子和保留或剪接另一个内含子的分子进行两两比较。发现了一个高度优选的去除顺序：内含子3 > 2 > 4 = 1。这一顺序并不代表从转录本一端到另一端的线性进展，也与剪接位点序列与共识序列的一致性或与U1小核RNA双工形成的计算能量无关。通过使用放线菌素D抑制RNA合成，估计了所有四个内含子剪接第一步的体内速率；内含子2、3和4的半衰期为6分钟。内含子1的剪接速度较慢，半衰期为12分钟。保留内含子1作为唯一内含子的大量RNA被输出到细胞质中。在这些实验过程中，我们还确定，在3'端形成完成之前，可能在新生转录本上剪切了内含子3，而不是内含子4。这一结果与多聚腺苷化是剪接3'-most内含子所必需的观点是一致的。我们采用了类似的策略来确定一个非常大的转录本中最后一个剪接的内含子，即中国仓鼠卵巢细胞内源性二氢叶酸还原酶（dhfr）基因（25 kb）。这里，1号内含子是最后一个被拼接的内含子。
Using a strategy based on reverse transcription and the polymerase chain reaction, we have determined the order of splicing of the four introns of the endogenous adenine phosphoribosyltransferase (aprt) gene in Chinese hamster ovary cells. The method involves a pairwise comparison of molecules that retain one intron and have either retained or spliced another intron(s). A highly preferred order of removal was found: intron 3 > 2 > 4 = 1. This order did not represent a linear progression from one end of the transcript to the other, nor did it correlate with the conformity of the splice site sequences to the consensus sequences or to the calculated energy of duplex formation with U1 small nuclear RNA. By using actinomycin D to inhibit RNA synthesis, the in vivo rate of the first step in splicing was estimated for all four introns; a half-life of 6 min was found for introns 2, 3, and 4. Intron 1 was spliced more slowly, with a 12-min half-life. A substantial amount of RNA that retained intron 1 as the sole intron was exported to the cytoplasm. In the course of these experiments, we also determined that intron 3, but not intron 4, is spliced before 3'-end formation is complete, probably on nascent transcripts. This result is consistent with the idea that polyadenylation is required for splicing of the 3'-most intron. We applied a similar strategy to determine the last intron to be spliced in a very large transcript, that of the endogenous dihydrofolate reductase (dhfr) gene in Chinese hamster ovary cells (25 kb). Here again, intron 1 was the last intron to be spliced.