We present here the first insights into the organization
of proteins on the RNA in the U5 snRNP of Saccharomyces
cerevisiae. Photo-crosslinking with uniformly labeled
U5 RNA in snRNPs reconstituted in vitro revealed five contacting
proteins, Prp8p, Snu114p, p30, p16, and p10, contact by
the three smaller proteins requiring an intact Sm site.
Site-specific crosslinking showed that Snu114p contacts
the 5′ side of internal loop 1, whereas Prp8p interacts
with five different regions of the 5′ stem-loop,
but not with the Sm site or 3′ stem-loop. Both internal
loops in the 5′ domain are essential for Prp8p to
associate with the snRNP, but the conserved loop 1 is not,
although this is the region to which Prp8p crosslinks most
strongly. The extensive contacts between Prp8p and the
5′ stem-loop of U5 RNA support the hypothesis that,
in spliceosomes, Prp8p stabilizes loop 1–exon interactions.
Moreover, data showing that Prp8p contacts the exons even
in the absence of loop 1 indicate that Prp8p may be the
principal anchoring factor for exons in the spliceosome.
This and the close proximity of the spliceosomal translocase,
Snu114p, to U5 loop 1 and Prp8p support and extend the
proposal that Snu114p mimics U5 loop 1 during a translocation
event in the spliceosome.