Edwards JM , Long J , de Moor CH , Emsley J , Searle MS .

BB/I011420/1 Biotechnology and Biological Sciences Research Council , BB/G001847/1 Biotechnology and Biological Sciences Research Council , BB_BB/G001847/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Full-length sequence of CELF1 showing RRM domains and linker sequences; alignment with the Δ(215–384) mutant RRM123 (a). RNA-binding sequence for CELF1 with three UGU sites separated by Ux and Uy spacers (b). Binding curves from ITC studies of CELF1 construct RRM123 with the RNA sequences EDEN11 (x = 1, y = 1) (GRE), EDEN-2U/4U (x = 2, y = 4) (c), and for EDEN-2U/1U (x = 2, y = 1) and EDEN-2U/HL (x = 2, y = 14) (d) (see sequences in Table 1). Total heat released is plotted as a function of the molar ratio of RNA to protein, and the non-linear least squares fit to the experimental data are shown as a solid line. Dissociation constants, binding stoichiometries and enthalpies of binding are shown in Table 2.
	Figure 2. 1H-15N TROSY spectra of the RRM123 construct at 800 MHz. Overlayed spectra of the unbound RRM123 (blue) with RRM123 complexes with EDEN11 (red) and EDEN-4U (green) at an RNA:protein ratio of 3:1. The region shown highlighted in (a) containing cross-peaks of some Gly and Cys residues is expanded in (b). Key residues from all three RRMs that show significant CSP effects on RNA binding are labelled. The proposed binding stoichiometry is shown schematically.
	Figure 3. NMR analysis for binding of RRM123 to the high affinity sequences EDEN-2U/4U and EDEN-2U/HL. (a) Overlayed portions of the 1H-15N-TROSY spectrum at 800 MHz of RRM123 (unbound in black) showing large CSPs for C61 (RRM1), C150 (RRM2) and G278 (RRM3) at a 1:1 binding ratio with the RNA sequences EDEN-2U/4U (blue) and EDEN-2U/HL, (red); (b) histogram of CSPs for RRM123 complex formation with EDEN-2U/4U with CSP effects >0.2 ppm labelled; (c) model of the RRM123 structure showing all CSPs >0.1 in red with residue side chains illustrated for CSPs > 0.2 ppm. Some residues broaden and disappear completely and are coloured in yellow; (d) RNA sequences coloured to match spectra in (a), with UGU-binding sites underlined.
	Figure 4. PRE effects in binding of RRM123 to spin-labelled EDEN-2U/4U substrates with MTSL covalently attached through 4-thiouridine (U*) at the 5′ or 3′ terminus. (a) Histogram showing PRE effects from 5′-U* across all three domains with the largest attenuation effects >0.7 clustered in RRM2. (b) PRE effects from 3′-U* with specific effects now evident in RRM3; (c) RNA sequence and model of the structure of 5′-U* labelled EDEN-2U/4U with RRM123 showing the location of the largest PRE effects. All residues with PREs >0.7 are shown in red. With the exception of Met18, all of these are in RRM2. Residues with PREs between 0.5 and 0.7 are shown in orange. The modified base to which the MTSL tag is attached (U*) is shown in blue. The RNA sequence used in the modeling is also shown with the three guanines highlighted on the structure; (d) PRE effects from 3′-U* labelled EDEN-2U/4U, with residues with PRE effects >0.8 shown in red (>0.7 in orange); (e) overlayed portions of the TROSY spectra of the complexes of RRM123 with 5′-labelled EDEN-2U/4U (black) and 3′-labelled EDEN-2U/4U (red) showing differentiation signal attenuation effects particularly for G176 (RRM2) and E244 (RRM3). The structure of MTSL modified 4-thiouridine (U*) is also shown.
	Figure 5. Structure and conformational flexibility of the RRM123 complex with EDEN-2U/4U. (a) Model refined from MD simulations showing the arrangement of the three RRMs in the order 2-1-3 from 5′ to 3′ along the RNA substrate. (b) The flexible 35-residue linker between RRM2 and RRM3 allows the C-terminal domain to fold back, as shown schematically. (c) Overlayed structures from the MD ensemble showing changes in orientation about the 8-residue RRM1-RRM2 linker sequence which allows RRM123 to adopt both compact and more extended conformations.
	Figure 6. (a) Structural model from MD simulations of the complex of the CELF1 construct RRM123 with the hairpin-loop mRNA EDEN-2U/HL; (b) schematic representation for clarity. The RNA hairpin is effective in co-localizing all three single-stranded UGU-binding motifs, with RRM123 binding across the base of the hairpin stem-loop. (c) Mfold prediction of secondary structure formation in part of the 3′-UTR of c-jun, which triggers deadenylation. UGU-binding sites (highlighted in red) are founded within an unstructured GU-rich loop and are co-localized through the formation of two hairpin structures.

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