Champion-Arnaud P, Gozani O, Palandjian L, Reed R
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1995 Oct;15(10):5750-6. doi: 10.1128/MCB.15.10.5750.
Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.
前体mRNA在E→A→B→C的逐步途径中组装成剪接体复合物。我们发现，后生动物分支点序列（BPS）中的突变对E复合物的形成没有明显影响，但会阻止A复合物的组装以及U2小核核糖核蛋白颗粒（snRNP）蛋白的紫外线交联。出乎意料的是，一种名为E的新型复合物会在含有BPS突变的前体mRNA上组装。与E复合物不同，E复合物在ATP存在的情况下会积累。在E复合物中与前体mRNA紧密结合的U1 snRNP和U2AF，在E复合物中并非紧密结合。重要的是，先前的研究表明，在正常前体mRNA上组装E复合物后，U1 snRNP和U2AF会从前体mRNA上变得不稳定。因此，我们的数据与一个模型一致，即在从E复合物到A复合物的转变中有两个步骤（E→E→A）。在第一步中，U1 snRNP和U2AF在ATP依赖、BPS独立的反应中变得不稳定。在第二步中，U2 snRNP的稳定结合发生在BPS依赖的反应中。
