| Splicing, the removal of non-protein-coding introns from pre-mRNA, is a critical step in eukaryotic gene expression. Splicing is catalyzed by the spliceosome, which is composed of five small nuclear RNAs (U1, U2, U4, U5, and U6) and over 100 proteins. To facilitate molecular dissection of the structure and function of U4, we have developed an in vitro assay for reconstitution of functional U4 snRNPs. Depletion of U4 strongly inhibited splicing, and subsequent addition of wild-type in vitro transcribed U4 allowed efficient recovery of splicing. Analysis of reconstitution by U4 3' truncation mutants showed that, while the Sm protein binding site was dispensable for splicing in vitro, shorter mutants lacking the 3' stem-loop were unable to reconstitute splicing. We showed that the 3' stem-loop was essential for formation of the yeast di-snRNP, but it was dispensable for subsequent steps of spliceosome assembly and splicing. |
