Freyer G A, O'Brien J P, Hurwitz J
Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York 10021.
J Biol Chem. 1989 Sep 5;264(25):14631-7.
The polypyrimidine tract, located at the 3' end of intron 1 of the adenovirus major late transcript, was studied for its role in splicing using cell-free preparations isolated from HeLa cells. A plasmid (pIz) was constructed in which seven purine bases were substituted for pyrimidine bases within the 14-nucleotide polypyrimidine sequence. Runoff transcripts extending to the middle of intron 2 were tested for their ability to support in vitro splicing. The efficiency of these reactions was compared with pre-mRNA transcripts made from the wild-type nonmutated plasmid (p1-2). Neither spliced products nor splicing intermediates were detected in reactions with the pIz pre-mRNA. The formation of the nucleoprotein complexes involved in splicing was examined with this altered pre-mRNA. No 55 S splicing complex was detected and only low levels of the 30 S presplicing complex formed (30-fold less than with wild-type pre-mRNA). However, when a longer runoff transcript was prepared from the polypyrimidine mutated plasmid pIz, spliced RNA was formed. This activity required specific downstream sequences, since transcripts produced from pIz which contained substituted downstream sequences were not spliced. Although intron 2 of the adenovirus major late transcript does not contain a discernible 3' polypyrimidine sequence, pre-mRNA (p2-3) containing this intron was efficiently spliced. However, when the 3' region of intron 2 was substituted for the polypyrimidine sequence of intron 1, the resulting pre-mRNA did not support efficient splicing in vitro. However, when the polypyrimidine sequence of intron 1 was substituted for the sequence at the 3' end of intron 2, efficient splicing occurred, and the rate of formation of splicing intermediates and the accumulation of nucleoprotein complexes was greater than with the wild-type pre-mRNA (p2-3).
利用从HeLa细胞中分离得到的无细胞制剂，研究了位于腺病毒主要晚期转录本内含子1 3'端的多嘧啶序列在剪接中的作用。构建了一个质粒（pIz），其中在14个核苷酸的多嘧啶序列内，7个嘌呤碱基取代了嘧啶碱基。对延伸到内含子2中部的连续转录本进行体外剪接能力测试。将这些反应的效率与野生型非突变质粒（p1-2）产生的前体mRNA转录本进行比较。在与pIz前体mRNA的反应中，未检测到剪接产物和剪接中间体。用这种改变后的前体mRNA检查参与剪接的核蛋白复合物的形成。未检测到55S剪接复合物，仅形成了低水平的30S剪接前复合物（比野生型前体mRNA少30倍）。然而，当从多嘧啶突变质粒pIz制备更长的连续转录本时，形成了剪接RNA。这种活性需要特定的下游序列，因为从含有取代下游序列的pIz产生的转录本未被剪接。尽管腺病毒主要晚期转录本的内含子2不包含可识别的3'多嘧啶序列，但含有该内含子的前体mRNA（p2-3）能有效剪接。然而，当内含子2的3'区域取代内含子1的多嘧啶序列时，产生的前体mRNA在体外不支持有效剪接。然而，当内含子1的多嘧啶序列取代内含子2 3'端的序列时，发生了有效剪接，并且剪接中间体的形成速率和核蛋白复合物的积累比野生型前体mRNA（p2-3）更大。
