Pre-messenger RNA splicing is a two-step process by which
introns are removed and exons joined together. In yeast,
the U5 snRNA loop 1 interacts with the 5′ exon before
the first step of splicing and with the 5′ and 3′
exons before the second step. In vitro studies revealed
that yeast U5 loop 1 is not required for the first step
of splicing but is essential for holding the 5′ and
3′ exons for ligation during the second step. It
is critical, therefore, that loop 1 contacts the 5′
exon before the first step of splicing to hold this exon
following cleavage from the pre-mRNA. At present it is
not known how U5 loop 1 is positioned on the 5′ exon
prior to the first step of splicing. To address this question,
we have used site-specific photoactivated crosslinking
in yeast spliceosomes to investigate the interaction of
U5 loop 1 with the pre-mRNA prior to the first step of
splicing. We have found that the highly conserved uridines
in loop 1 make ATP-dependent contacts with an approximately
8-nt region at the 5′ splice site that includes the
invariant GU. These interactions are dependent on functional
U2 and U6 snRNAs. Our results support a model where U5
snRNA loop 1 interacts with the 5′ exon in two steps
during its targeting to the 5′ splice site.