Effects of Cwc2 mutagenesis on its splicing and RNA-binding activity. (A) Western blot analysis of yeast splicing extracts carrying TAP-tagged Cwc2 before depletion (lane 1) and after depletion of Cwc2 (lane 2). (B) Uniformly radiolabelled M3-actin pre-mRNA (0.4 nM) was incubated in 20% yeast whole-cell extract, which was either mock- (lanes 1 and 2) or Cwc2-depleted (lanes 3–10), under standard splicing conditions. Recombinant WT Cwc2 and mutant proteins indicated above each lane were then added to a final concentration of 2 μM (lanes 4–10). The splicing reactions were incubated at 23°C for the indicated time points. RNA was analysed on an 8% urea–polyacrylamide gel and visualized by autoradiography. The positions of the pre-mRNA, the splicing intermediates and products are indicated on the left. The intensities of the mRNA product signals were quantified using Quantity One software (Bio-Rad). The signal of the Cwc2–TAP extract (lane 2) was used as 100%, and relative to it the amount of mRNA produced was calculated. ΔCwc2 extract shows a residual level of mRNA of 16% (lane 3) compared with 109% for ΔCwc2 extract supplemented with WT Cwc2 (lane 4), 97% with Cwc2ΔC (lane 5), 16% with RRM (lane 6), 50% with Y89A (lane 7), 21% with Y120A (lane 8), 58% with K132A (lane 9) and 69% with F130A (lane 10). The mRNA level in the lane 3 was set as a background (remaining splicing activity of the Cwc2-depleted extract) and the restoration of splicing activity due to complementation with recombinant Cwc2 or its mutants was calculated as a difference between mRNA signals in the corresponding lane and lane 3. (C) Electrophoretic mobility shift analysis of the interaction of recombinant Cwc2 protein and its truncations with yeast U6 snRNA. Equal amounts of [³²P]-5′-labelled U6 snRNA were incubated in the presence of increasing concentrations of purified full-length Cwc2 (wt), its stable GluC endoproteolytic fragment (ΔC) or its RRM. Resulting RNP complexes and free U6 RNA were resolved by native 6% PAGE. The identity and concentrations of the proteins used are indicated above the gel. Migration positions of RNA–protein complexes and unbound U6 snRNA is shown on the right. Asterisks mark a slower migrating band of U6 snRNA observed in protein-free samples.

Structural overview. (A) Stereo ribbon plot of the Cwc2 structure. Secondary structure elements and termini are labelled. Red, ZnF domain; blue, Torus domain; green, RRM domain; yellow, variable insertions; orange, connector element. Aromatic residues from the two canonical RNP motifs are shown as magenta sticks. (B) Intimate association between the ZnF and the Torus domain. The residues coordinating the zinc atom are depicted as sticks and labelled (left). The close-up view highlights conserved aromatic residues that stabilize the Torus domain around the ZnF (right). (C) Interaction of the RRM domain with the ZnF and the Torus domain (left). Close-up view highlighting RRM contacts with the ZnF and Torus domains. Highly conserved residues are depicted as sticks and labelled (right). The relative orientations versus the position from (A) is indicated.

Domain composition and sequence alignment of Cwc2. (A) Schematic depiction of domains and structural motifs in Cwc2. (B) Multiple sequence alignment of Cwc2 orthologues. S.c.—S. cerevisiae; K.l.—Kluyveromyces lactis; Y.l.—Yarrowia lipolytica; S.p.—Schizosaccharomyces pombe; C.a.—Candida albicans; C.n.—Cryptococcus neoformans; H.s.—Homo sapiens. Darker background corresponds to higher sequence conservation. Icons above indicate secondary structure elements from the Cwc2 crystal structure. Magenta background indicates the RNA-crosslinked sites. The triangles indicate mutated amino acids tested in splicing complementation assays. (C) Comparison of consensus RNP1 and RNP2 sequences (Maris et al, 2005) in Cwc2 and U1A, respectively. The amino-acid residues important for RNA binding are depicted in red. X represents any amino acid.

Comparison of the Cwc2 TlS11d ZnF. (A) Superposition of a CCCH-type ZnF domain of TIS11d on Cwc2. Cwc2 residues potentially involved in binding RNA and the four residues coordinated by zinc in the two proteins are shown and labelled. (B) Recognition of an AU-rich RNA element by the ZnF domain of TIS11d (pdb 1RGO) (Hudson et al, 2004), in the same orientation. The aromatic residues from TIS11d involved in stacking interactions with the bases are depicted as sticks.

Mapping of the RNA-crosslinked sites on the Cwc2 surface. (A) Electrostatic potential (B) surface conservation. Cyan—variable sequences. The molecule is shown in the same orientation as in Figure 2A. The residues crosslinked to RNA are outlined with dashed lines and labelled. The shallow depression is contoured with continuous lines. (C) Electrophoretic mobility shift analysis of the interaction of recombinant Cwc2 mutants with yeast U6 snRNA. Description as in Figure 1C. The amino acids mutated are shown above the gel.

Notable similarities with other RRM domains. (A) Comparative view of Cwc2 (left) and the RRM domain of U1A in complex with its cognate RNA (right; Oubridge et al, 1994). Three U1A residues that contact its cognate RNA (right) and residues located in equivalent positions in Cwc2 (left) are depicted as magenta sticks. The Cwc2 residues crosslinked to RNA are marked with black arrows. The ZnF–Torus module is shadowed for the sake of clarity. (B) Comparative view of the connector-RRM (orange–green) region from Cwc2 (left) and the topologically equivalent region of CBP20 in complex with a cap analogue (black; pdb 1H2T, left). Other regions of the two protein molecules have been omitted for clarity sake. Aromatic residues of CBP20 that form the binding pocket of the cap (right) and Cwc2 residues that might cooperate in a similar manner (left) are depicted as magenta sticks.