SECONDARY STRUCTURE OF THE YEAST SACCHAROMYCES-CEREVISIAE PRE-U3A SNORNA AND ITS IMPLICATION FOR SPLICING EFFICIENCY

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical an d enzymatic methods to probe the RNA secondary structure and site-dire cted mutagenesis, we established the complete secondary structure of t he U3A snoRNA precursor. This is the first determination of the comple te secondary structure of an RNA spliced in a spliceosome. The peculia r cruciform structure of the U3A snoRNA 3'-terminal region is formed i n the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins. The intron forms a highly folded stru cture with a long central stem-loop structure that brings the 5' box a nd the branch site together. This is in agreement with the idea that s econdary structure interactions are necessary for efficient splicing o f long introns in yeast. The 3' splice site is in a bulged loop and th e branch site sequence is single-stranded. Surprisingly, the 5' splice site is involved in a 6-base pair interaction. We used in vitro splic ing experiments to show that, despite a noncanonical branch site seque nce and a base paired 5' splice site, transcripts that mimic the authe ntic pre-U3A snoRNA are spliced very efficiently in vitro. Sequesterin g the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence. Analysis of spliceosomal comp lex formation revealed a cumulative negative effect of a base pair int eraction at the 5' splice site and of a deviation to the consensus seq uence at the branch site on the efficiency of spliceosome formation in vitro.