Edwards J , Malaurie E , Kondrashov A , Long J , de Moor CH , Searle MS , Emsley J .

076179 Wellcome Trust , BB/F013663/1 Biotechnology and Biological Sciences Research Council , BB/G001847/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Schematic representation of the protein constructs used in this study and an indication of domain boundaries. The position of the RRMs are shown shaded. Residue Ser28 represents the proposed Akt phosporylation site in RRM1.
	Figure 2. The first 187 amino acids of CELF1 are sufficient for specific binding to natural and synthetic binding sites. (a) UV-crosslinking of BSA control (C), full length CELF1 (FL), T353 (T) with radioactive maskin 3′-UTR (Msk), a truncated cyclinB1 3′-UTR (xxsB1) and a Control RNA (Bluescript polylinker transcript). Sizes of crosslinked bands are indicated. (b) UV-Crosslinking competition assay. The maskin 3′-UTR was competed with synthetic EDEN15. Radioactive control RNA (Bluescript: BS) or maskin 3′-UTR with EDEN15 in the indicated molar ratios were incubated with T187 and covalently crosslinked using by UV light. (c) Gel retardation assay with T187 EDEN15, EDEN11 and EDEN7, as well as with the AU rich element (ARE15) incubated in buffer A. An amount of 50 nM of RNA end labelled with γ-P32-ATP was incubated with increasing concentration of t187 protein, ratio RNA: protein from 1:0 to 1:100. Slender arrows indicate free RNA, thick arrows complexes induced by the protein. (d) Gel retardation of 45 nM GUCU15 with increasing concentrations of T187 in buffer B. (e) Gel retardation of 45 nM EDEN19 with increasing concentrations of T187 in buffer B.
	Figure 3. CELF1 RNA complex gel filtration profiles. (a) Plot of UV absorbance versus elution volume for T187 protein (blue) with RNA EDEN15 (red) mixed at two different molar ratios, 1:0.25 (pink); 1:1 (green), 1:2 (turquoise). (b) CELF1 RRM1 RNA complex gel filtration profiles. In blue RRM1 and in red EDEN7 alone. Different ratios RRM1:EDEN7: 1:0.25 (pink); 1:0.5 (orange); 1:1 (green) and 1:2 (turquoise).
	Figure 4. 2D 1H/15N-TROSY NMR spectra of T187 (a) and overlayed spectra of RRM1 (red) and RRM2 (blue) in (b) showing that the longer construct is well represented by the sum of the spectra of the individual domains, except for a few perturbations at the domain boundaries. Spectra were collected at 298 K in 25 mM phosphate buffer, 50 mM NaCl, 10% D2O (v/v), pH 7.0 with protein sample concentrations in the range 400–500 µM.
	Figure 5. NMR CSP plots for the binding of EDEN 7 (UGUUUGU) and CUGCUG to the two isolated RRMs. In (a) and (b), EDEN7 binding to RRM1 is illustrated, and in (c) and (d), binding of CUGCUG to RRM1 and RRM2. In each case an arbitrary cut-off of 0.1 ppm is shown by the dotted line with residues showing CSPs > 0.1 ppm individually labelled. In addition, proline residues are marked with a black dot and residues which broaden and disappear during the titration are marked with an arrow head. Along the top of the figure the relative position of the protein secondary structure is indicated.
	Figure 6. Structural representation of the CSPs mapped to the surface of the RRM1 (a), RRM2 (b) and T187 (c, d) for the RNA indicated. The magnitude of the CSPs is shown on a red to grey scale (largest perturbations shown in brightest red). Residues which broaden and disappear are marked in yellow, as particularly evident for the RRM1 domain of T187 on binding EDEN7.
	Figure 7. NMR CSP plots for the binding of EDEN15 (a) and CUG15 (b) to T187. In each case an arbitrary cut-off of 0.1 ppm is shown by the dotted line with residues showing CSPs > 0.1 ppm individually labelled. In addition, residues which broaden and disappear during the titration are marked with an arrow head. No assignments were obtained for proline residues and these are marked with a black dot. Along the top of the figure the relative position of the protein secondary structure is indicated with the domain boundary between RRM1 and RRM2 shown at residue 110. Representative portions of the 1H/15N TROSY spectra from the two titrations are shown in (c) and (d). In particular, residue C150 in RRM2 is perturbed by the binding of EDEN15 (c), but not by CUG15 (d). Five TROSY spectra are overlayed in each case representing protein:RNA ratios in the range 1:0–1:1. (e) Similar overlayed portions of the 1H/15N TROSY spectrum of T187 showing the limiting CSPs for Cys61 (RRM1) and Cys150 (RRM2) at binding saturation with a series of related RNA sequences containing different U spacers UGU(U)xUGU, where x = 1–7. (f) Histogram plot showing the CSP data from (e) for Cys61 and Cys150 with the CSPs for RRM1 reaching a plateau between x = 2–5; longer U spacers (x = 6 and 7), or a very short spacer (x = 1) result in a reduction in binding affinity for RRM2 but little difference in the shift of Cys61 in RRM1. (g) Binding isotherms at 298 K from ITC studies of T187 with UGU(U)xUGU sequences where x = 1, 2 and 6. The shortest sequence (x = 1) shows a biphasic binding behaviour whereas the two other sequences (x = 2 and 4) fit to a 1:1 binding model.
	Figure 8. Schematic representation of the binding of RRM1, RRM2 and T187 to RNA substrates. (a) EDEN7 is able to accommodate two RRM1s on adjacent UGU sites, but only a single RRM2 can bind to the same sequence, suggesting specificity for a UGUU binding site. (b) EDEN7 is too short to bind the two RRMs of T187 in tandem, instead we observe an interaction through RRM1 which could be at either of the two UGU sites (one shown). (c) Binding of T187 to both sites of the consensus GRE sequence (EDEN11) with RRM2 bound at the 5′-terminal UGUU site. The spacer sequence of 5 nt between the tandem UGU sites results in high affinity binding. (d) Possible interaction of full-length CELF1 with a GU-rich substrate with RRM1 and RRM2 bound as for the consensus sequence in (c); the long linker between RRM2 and RRM3 permits considerable conformational flexibility in binding a third UGU site up or down stream (position of arrows), or even to the looped-out spacer sequence between the RRM1 and RRM2 binding sites.

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