Ge H, Noble J, Colgan J, Manley J L
Department of Biological Sciences, Columbia University, New York, NY 10027.
Proc Natl Acad Sci U S A. 1990 May;87(9):3338-42. doi: 10.1073/pnas.87.9.3338.
We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron.
我们已经在体外研究了多瘤病毒早期区域前体mRNA的剪接。这种RNA在体内会进行可变剪接，以产生编码大、中（MTAg）、小（StAg）肿瘤抗原的mRNA。我们主要感兴趣的是了解48个核苷酸的StAg内含子是如何被切除的，因为这个内含子的长度明显小于哺乳动物内含子所确定的明显最小值。尽管在体外检测到了所有三种剪接产物，但对StAg剪接途径和序列要求的表征表明，剪接因子以前所未有的方式与前体RNA相互作用，从而催化该内含子的切除。具体而言，StAg剪接使用两个套索分支点中的任何一个，其中一个仅位于距3'剪接位点4个核苷酸处。此外，StAg剪接绝对要求位于StAg 3'剪接位点下游14个核苷酸处的可变MTAg 3'剪接位点保持完整。增加或减少MTAg嘧啶延伸质量的插入突变会增强或抑制StAg以及MTAg剪接，并且MTAg AG剪接受体中的单碱基变化会完全阻断这两种剪接。这些结果证明了两个3'剪接位点相互协作以切除单个内含子的能力。
