SPLICEOSOME ASSEMBLY IN YEAST
SPLICEOSOME ASSEMBLY IN YEAST
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DOI：
10.1101/gad.1.9.1014
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发表时间：
1987-11-01
影响因子：
10.5
通讯作者：
ABELSON, J
中科院分区：
文献类型：
--
作者：
CHENG, SC;ABELSON, J
mRNA的前体通过组装成复杂的多亚基结构（剪接体）而成为剪接的底物。为了研究酵母剪接体的组装，在非变性聚丙烯酰胺凝胶上通过电泳分离中间复合物。观察到四种剪接依赖性复合物，A1，A2-1，A2-2和B。这些复合物的组装顺序被确定为B-A2-1-A1-A2-2。通过加入5 mM EDTA或通过在热灭活的rna 2提取物中进行组装过程，可以在复合物A1处阻断组装过程。通过与5种酵母snRNA的探针杂交来确定复合物的snRNA组成。snR 14（U4）仅见于复合物A2-1中，snR 6（U6）和snR 7（U 5）见于复合物A1、A2-1和A2-2中，而snR 20（U2）见于所有四种复合物中。snR 19（U1）不存在于任何复合物中。与这些探针的杂交也用于检测酵母剪接提取物中存在的snRNP。我们发现snR 6、snR 7和snR 14共同存在于一个大的复合物中。该复合物经历ATP依赖性解离，得到snR 7和snR 6-snR 14复合物。snR 19和snR 20存在于不同的RNP中，但它们的移动性不受ATP的影响。提出了一种剪接体组装的机制。
Precursors to mRNA become substrates for splicing by being assembled into a complex multisubunit structure, the spliceosome. To study the assembly of the yeast spliceosome, intermediate complexes were separated by electrophoresis on nondenaturing polyacrylamide gels. Four splicing-dependent complexes, A1, A2-1, A2-2, and B, were observed. The order of assembly of these complexes was determined to be B----A2-1----A1----A2-2. The assembly process can be blocked at complex A1 by addition of 5 mM EDTA or by carrying out the assembly process in heat-inactivated rna2 extracts. The snRNA composition of the complexes was determined by hybridization with probes for five yeast snRNAs. snR14 (U4) was only found in complex A2-1, snR6 (U6) and snR7 (U5) were in complexes A1, A2-1, and A2-2, whereas snR20 (U2) was in all four of the complexes. snR19 (U1) was not present in any of the complexes. Hybridization with these probes was also employed to detect snRNPs present in yeast splicing extracts. We found that snR6, snR7, and snR14 were present together in a large complex. This complex underwent an ATP-dependent dissociation to give snR7 and snR6-snR14 complexes. snR19 and snR20 are present in distinct RNPs but the mobility of these is not affected by ATP. A mechanism for spliceosome assembly is proposed.