The purification by a series of FPLC chromatographic steps and other methods of factor(s) functionally complementing pre-formed splicing complexing is proposed. Two assay systems have been developed that can be used to follow the complementing activity(ies) in fractionated HeLa cell nuclear splicing extracts. The first system?consists of operationally defined nuclear matrix that contains in vivo assembled complexes involved in nuclear pre- mRNA splicing. The splicing of matrix-associated globin pre-mRNA by in vitro complementation with factors in fractions of the splicing extract is monitored by high-resolution Northern analysis. The second system employs in vitro assembled, complementable SP6 spliceosomes, and electrophoretic analysis of the labeled products. The second system yields results faster, but the first system provides a reliable criterion for functionality because it simulates the in vivo situation. Depending on the method of preparation, each system also exists in the form of self-splicing complexes. Experiments are proposed, based on the behavior of the two versions of each system, to characterize the features of the complementing factor(s) and the biochemical parameters that affect their functionality. Cloning of the gene(s) encoding the complementing factor(s) is proposed as a long-term goal.
| Parent, A; Wilson, R C; Zeitlin, S et al. (1989) Complementation of in vitro-assembled spliceosomes. J Mol Biol 209:379-92 |
