Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site

Abstract

In the yeast Saccharomyces cerevisiae the 5' and 3' splice junctions and the internal branch acceptor site (TACTAAC box) are highly conserved intron elements. Analyses of mutants have demonstrated the importance of each of these elements in the splicing process. In the present report we show by three different analytical approaches (splicing-dependent beta-galactosidase expression, in vitro splicing assays and in vivo RNA analyses) that at least two of these elements (the TACTAAC and 3' splice signals) also have to fulfill certain spacing requirements to allow efficient splicing to occur. In particular, the spacing of the 3' splice site from the 2'-5' branch site is a critical factor in determining the efficiency for completion of the final reactions of splicing, intron release and exon-exon joining. Whereas insertions within this region have little or no effect on the first reactions in splicing (the 5' cleavage and 2'-5' branch formation), they dramatically affect the efficiency of the final reactions. In contrast, a 15-base deletion between these two sites has no detectable effect on splicing efficiency. We also show that the 5' cleavage and branch formation can take place, albeit inefficiently, in transcripts in which all of the yeast sequences downstream of the branch site have been replaced by Escherichia coli sequences. We conclude from these studies that, in yeast, the 5' and 3' splice sites are recognized independently from one another, but always in conjunction with the TACTAAC signal.