Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the TACTAAC box in vivo and in vitro. In contrast, in the HeLa cell extract, the yeast pre-mRNA is accurately spliced, but the RNA lariats contain RNA branches located significantly closer to the 3' splice site than the TACTAAC box. In yeast, mutant pre-mRNAs that lack the TACTAAC box are not spliced in vivo or in vitro. However, these same mutant pre-mRNAs are accurately spliced in the HeLa cell extract. Therefore, although pre-mRNA splicing in yeast and higher eukaryotes proceeds by the same basic pathway, there are substantial differences in the specificity of the biochemical components that mediate the formation of the RNA processing products.