FUNCTIONAL-ANALYSIS OF THE U5 SNRNA LOOP-1 IN THE 2ND CATALYTIC STEP OF YEAST PRE-MESSENGER-RNA SPLICING

The U5 snRNA loop 1 interacts with the 5' exon before the first step o f pre-mRNA splicing and with the 5' and 3' exons following the first s tep. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critica l for the second step in yeast spliceosomes. We systematically made de letion and insertion mutations in loop 1 then monitored splicing activ ity and loop-exon interactions by cross-linking. Single nucleotide del etions or insertions in loop 1 permitted both steps of splicing. Large r insertions or deletions allowed the first step but progressively inh ibited the second step. Analysis of selected loop 1 insertions and del etions by cross-linking revealed that inhibition of the second catalyt ic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of t he exons for the second catalytic step of splicing and that the 3' exo n is positioned on loop 1 independently of the 5' exon.