The structure of the CstF-77 homodimer provides insights into CstF assembly

Abstract

The cleavage stimulation factor (CstF) is essential for the first step of poly(A) tail formation at the 3' ends of mRNAs. This heterotrimeric complex is built around the 77-kDa protein bridging both CstF-64 and CstF-50 subunits. We have solved the crystal structure of the 77-kDa protein from Encephalitozoon cuniculi at a resolution of 2 A. The structure folds around 11 Half-a-TPR repeats defining two domains. The crystal structure reveals a tight homodimer exposing phylogenetically conserved areas for interaction with protein partners. Mapping experiments identify the C-terminal region of Rna14p, the yeast counterpart of CstF-77, as the docking domain for Rna15p, the yeast CstF-64 homologue.

Figure 1.

Structure of CstF-77 homodimer. (a) CstF-77 is built entirely of α-helices belonging to the HAT-repeat family (labelled α1–α23), with the exception of disordered residues (dotted lines). Only one of the two monomers is coloured from blue to red (N to C terminus). The second monomer is shown in grey. HAT repeats are indicated. (b) Schematic representation of CstF-77 homodimer. The two views are related by a 90° rotation as indicated. (c) Stereoview of CstF-77 homodimer.

Figure 2.

Superimposition of murine and E. cuniculi CstF-77 orthologues. The HAT-N (a) and HAT-C domains (b) of the murine CstF-77 (red) were individually superimposed on the N- and middle domain of CstF-77 from E. cuniculi (grey).

Figure 3.

Sequence alignment CstF-77 homologues. The polypeptide sequence of CstF-77 from E. cuniculi, H. sapiens, D. melanogaster, C. elegans, X. laevis, A. thalina and S. cerevisiae were aligned with PipeAlign (47). Residues are coloured in red when >75% identity is reached. Secondary structures are shown for each monomer on top of the sequences. Dotted lines correspond to sequences for which no interpretable electron density was seen. HAT repeats are boxed in beige. The location of the mutated amino acid in rna14-1, rna14-5 and Drosophila Su(f) R-9-18 mutant are boxed in red.

Figure 4.

CstF-77 homodimer exposes conserved surfaces for interaction with partners. (a) and (b) Residue conservation has been calculated with Consurf from a sequence alignment including CstF-77 homologues. Conservation ranges from white for non-conserved residues, to red for absolutely conserved residues. The two orientations of the complex are related by a 90° rotation in the y- and z-axis. The border of the dimer interface is shown as a solid grey line on both panels.

Figure 5.

Mapping Rna14p–Rna15p interaction. (a) Pull-down experiments were performed between Rna15p and His-tagged Rna14p deletion mutants. Proteins were revealed with polyclonal antibodies to Rna15p and monoclonal antibodies directed against the His-tag. T, total extract; B, bound fraction. (b) Rna14p deletion constructs used in pull-down experiments. ‘+’ and ‘–’ corresponds to interaction and no interaction, respectively.