DC mutations and Shq1 interaction. (A) Sequence alignment around the Cbf5 PUA domain. The aligned Cbf5 sequences are from S. cerevisiae (Sc), A. thaliana (At), C. elegans (Ce), D. melanogaster (Dm), H. sapiens (Hs) and P. furiosus (Pf). Residues that are conserved in 100 and 80% of the five eukaryotic sequences are shaded by black and grey, respectively. Pf residues that match the conserved eukaryotic residues are shaded. The observed secondary structure elements are indicated with the NTE and CTE coloured in magenta. DC mutations are labelled and denoted by orange dots below the human dyskerin sequence (X for deletion). Yeast Cbf5 residues with solvent accessible surface buried by at least 30 and 10 Å2 due to Shq1 association are marked by blue solid and open circles, respectively. Pf Cbf5 residues buried by H/ACA RNA are marked by red circles in a similar manner. (B) DC mutations mapped to the PUA domain and the C-terminal extension of the yeast Cbf5–Shq1 structure (cross-eye stereo-view). The DC mutation sites are indicated by orange spheres for Cα atoms and sticks, and labelled with the residue number of yeast Cbf5. For the purposes of illustration, residues 5–11 not observed in the Shq1 complex structure are modelled as in the CNG structure. (C) Interaction between Shq1 and yeast Cbf5 with DC mutations. The Cbf5 DC mutant complexes co-purified with either Nop10 (CN) or Nop10 and Gar1 (CNG) were incubated with a 4-fold excess of Shq1 and pulled down with Ni beads. The input panel contained 5% of the material used for the pull-down. Only the portion of gel containing Cbf5 and Shq1 is shown. Δ354 and ΔN15 contains Cbf5 residues 1–354 and 16–394, respectively.