Zeitlin S, Wilson R C, Efstratiadis A
Department of Genetics and Development, Columbia University, New York 10032.
J Cell Biol. 1989 Mar;108(3):765-77. doi: 10.1083/jcb.108.3.765.
We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.
我们使用了一种体内系统，该系统可产生可检测量的特定前体mRNA，以研究剪接与一种操作定义的核基质制剂（NM）之间的关系。当通过用大约生理浓度的KCl（0.1M）提取经DNA酶I处理的细胞核来制备NM时，一部分与NM相关的前体可以在ATP和Mg2+存在且无剪接提取物的情况下在体外进行剪接（“自主剪接”）。我们提出，这种不表现出时间滞后且在5分钟内完成一半的自主反应，发生在完全组装的、与基质结合的核糖核蛋白复合物（体内剪接体）中。用浓度大于0.4M的KCl提取NM会消除自主剪接，但会留下可以用HeLa细胞核提取物进行剪接互补的预组装复合物。剪接互补因子代表存在于核提取物以及细胞质S100组分中的一种或多种活性，相对耐热，不含RNA成分，并且在0.1M KCl中不与DEAE-琼脂糖结合。它在细胞核中以两种形式存在；与自主剪接体结合以及在核质中游离。本文描述并讨论了互补反应的生化特征以及两个剪接步骤可逆解偶联的条件。
