ELECTROPHORETIC SEPARATION OF COMPLEXES INVOLVED IN THE SPLICING OF PRECURSORS TO MESSENGER-RNAS
ELECTROPHORETIC SEPARATION OF COMPLEXES INVOLVED IN THE SPLICING OF PRECURSORS TO MESSENGER-RNAS
复制标题
DOI：
10.1016/0092-8674(86)90066-8
复制
发表时间：
1986-09-12
期刊：
影响因子：
64.5
通讯作者：
SHARP, PA
中科院分区：
文献类型：
--
作者：
KONARSKA, MM;SHARP, PA
在天然低百分比聚丙烯酰胺凝胶上电泳分析了剪接配合物。两种不同的抗肝素复合物，A和B，特异性地组装在含有真实的5‘和3’剪接位点的RNA前体上。这个组装是atp依赖的。动力学实验表明，配合物A随着时间的推移转变为更大、迁移速度更慢的配合物B。凝胶电泳检测到的配合物A和B分别对应于25S和35S的物质沉积。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通道，使其不被RNAase T1消化。通过Northern杂交分析，复合物A和B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的内源性大复合物。
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.