Konarska M M, Sharp P A
Cell. 1986 Sep 12;46(6):845-55. doi: 10.1016/0092-8674(86)90066-8.
Splicing complexes were analyzed by electrophoresis on a native low-percentage polyacrylamide gel. Two distinct heparin-resistant complexes, A and B, are assembled specifically on an RNA precursor containing authentic 5' and 3' splice sites. This assembly is ATP-dependent. Kinetic experiments suggest that complex A is converted with time to a larger, slower migrating complex B. Complexes A and B detected by gel electrophoresis correspond to material sedimenting at 25S and 35S, respectively. Substrate RNA containing only the 3' splice site is capable of forming the smaller complex A but not complex B. Complex A protects sequences upstream of the 3' splice site, encompassing the branch site and polypyrimidine tract from digestion by RNAase T1. U2 snRNA, but not U1 snRNA was detected in both complexes A and B by Northern hybridization analysis. Interestingly, an endogenous large complex containing U2 snRNP could be detected in nuclear extracts.
通过在天然低百分比聚丙烯酰胺凝胶上进行电泳分析剪接复合体。两种不同的抗肝素复合体A和B，特异性地组装在含有真实5'和3'剪接位点的RNA前体上。这种组装是ATP依赖性的。动力学实验表明，复合体A随时间转化为更大、迁移更慢的复合体B。通过凝胶电泳检测到的复合体A和B分别对应于在25S和35S沉降的物质。仅含有3'剪接位点的底物RNA能够形成较小的复合体A，但不能形成复合体B。复合体A保护3'剪接位点上游的序列，包括分支位点和多嘧啶序列不被RNA酶T1消化。通过Northern杂交分析在复合体A和B中均检测到U2 snRNA，但未检测到U1 snRNA。有趣的是，在核提取物中可以检测到含有U2 snRNP的内源性大复合体。
