Abstract
This chapter examines yeast pre-mRNA sequence contribution to splicing efficiency. A primer complementary to intron sequences, RB27 can be used to determine the relative abundance of premRNA and lariat molecules. On addition of a small, splicing-competent substrate and ATP to a yeast whole cell extract, the time-dependent appearance of three splicing complexes could be visualized. Presumably the carrier RNA eliminates nonspecific and adventitious binding of proteins to the spliceosome complexes, thereby increasing the electrophoretic mobility of the complexes and improving resolution. The modified pre-mRNA is then added to an in vitro assembly reaction, and the splicing complexes are resolved on native polyacrylamide gels. The expectation is that the assembly process should be sensitive to chemical modification at nucleotide positions required for the formation of spliceosomes. If a particular modification is extremely deleterious to assembly it should be absent from the complex-derived RNA. The DNA oligonucleotides should be size-selected by HPLC chromatography or polyacrylamide gel electrophoresis prior to labeling. This precaution removes the shorter, incompletely synthesized molecules that compete for labeling and may lead to background problems during primer extension.