The assembly of the U4 and U6 snRNPs into the U4/U6 di-snRNP
is necessary for pre-mRNA splicing, and in Saccharomyces
cerevisiae requires the splicing factor Prp24. We have
identified a family of Prp24 homologs that includes the human
protein SART3/p110nrb, which had been identified
previously as a surface antigen in several cancers. Sequence
conservation among the Prp24 homologs reveals the existence
of a fourth previously unidentified RNA recognition motif (RRM)
in Prp24, which we demonstrate is necessary for growth of budding
yeast at 37 °C. The family is also characterized by a highly
conserved 12-amino-acid motif at the extreme C terminus. Deletion
of this motif in Prp24 causes a cold-sensitive growth phenotype
and a decrease in base-paired U4/U6 levels in vivo. The mutant
protein also has a reduced association with U6 snRNA in extract,
and is unable to interact with the U6 Lsm proteins by two-hybrid
assay. In vitro annealing assays demonstrate that deletion of
the motif causes a defect in U4/U6 formation by reducing binding
of Prp24 to its substrate. We conclude that the conserved
C-terminal motif of Prp24 interacts with the Lsm proteins to
promote U4/U6 formation.