Furdon P J, Kole R
Proc Natl Acad Sci U S A. 1986 Feb;83(4):927-31. doi: 10.1073/pnas.83.4.927.
Human beta-globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.
在HeLa细胞核提取物中对在第二个外显子或第一个内含子中截短的人β-珠蛋白mRNA进行了体外加工。含有短至53个核苷酸的第二个外显子片段的转录本能够高效剪接，而在3'剪接位点下游截短24或14个核苷酸的转录本即使能剪接也是低效的。然而，所有这些转录本在5'剪接位点都能高效且准确地切割。相比之下，在第一个内含子中、3'剪接位点上游54个核苷酸处截短的RNA根本不进行加工。这些发现表明，5'剪接位点的切割和随后的剪接步骤——即3'剪接位点处的切割和外显子连接——不一定是偶联的。抗Sm血清抑制完全剪接反应和5'剪接位点处的切割，表明某些核糖核蛋白颗粒参与了切割反应。5'剪接位点处的切割需要ATP和Mg2+，其浓度与完全剪接反应所需的浓度相似。
