Dynamic rearrangement of RNA structure is crucial
for intron recognition and formation of the catalytic core
during pre-mRNA splicing. Three of the splicing factors
that contain sequence motifs characteristic of the DExD/DExH-box
family of RNA-dependent ATPases (Prp16, Prp22, and the
human homologue of Brr2) recently have been shown to unwind
RNA duplexes in vitro, providing biochemical evidence that
they may direct structural rearrangements on the spliceosome.
Notably, however, the unwinding activity of these proteins
is sequence nonspecific, raising the question of how their
functional specificity is determined. Because the highly
conserved DExD/DExH-box domain in these proteins is typically
flanked by one or more nonconserved domains, we have tested
the hypothesis that the nonconserved regions of Prp16 determine
the functional specificity of the protein. We found that
the nonconserved N-terminal domain of Prp16 is (1) essential
for viability, (2) required for the nuclear localization
of Prp16, and (3) capable of binding to the spliceosome
specifically at the step of Prp16 function. Moreover, this
domain can interact with the rest of the protein to allow
trans-complementation. Based on these results,
we propose that the spliceosomal target of the unwinding
activity of Prp16, and possibly other DExD/DExH-box splicing
factors as well, is defined by factors that specifically
interact with the nonconserved domains of the protein.