Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs

Abstract

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.