PTB-associated splicing factor (PSF) has been implicated in
both early and late steps of pre-mRNA splicing, but its exact
role in this process remains unclear. Here we show that PSF
interacts with p54nrb, a highly related protein first
identified based on cross-reactivity to antibodies against the
yeast second-step splicing factor Prp18. We performed RNA-binding
experiments to determine the preferred RNA-binding sequences
for PSF and p54nrb, both individually and in
combination. In all cases, iterative selection assays identified
a purine-rich sequence located on the 3′ side of U5 snRNA
stem 1b. Filter-binding assays and RNA affinity selection
experiments demonstrated that PSF and p54nrb bind
U5 snRNA with both the sequence and structure of stem 1b
contributing to binding specificity. Sedimentation analyses
show that both proteins associate with spliceosomes and with
U4/U6.U5 tri-snPNP.