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.

Audic, Embryo deadenylation element-dependent deadenylation is enhanced by a cis element containing AUU repeats. 1998, Pubmed, Xenbase

Audic, Embryo deadenylation element-dependent deadenylation is enhanced by a cis element containing AUU repeats. 1998, Pubmed , Xenbase
Baldwin, Epidermal growth factor receptor stimulation activates the RNA binding protein CUG-BP1 and increases expression of C/EBPbeta-LIP in mammary epithelial cells. 2004, Pubmed
Barreau, Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions. 2006, Pubmed
Barreau, Protein expression is increased by a class III AU-rich element and tethered CUG-BP1. 2006, Pubmed
Beisang, Regulation of CUG-binding protein 1 (CUGBP1) binding to target transcripts upon T cell activation. 2012, Pubmed
Bonnet-Corven, An analysis of the sequence requirements of EDEN-BP for specific RNA binding. 2002, Pubmed , Xenbase
Castle, Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines. 2008, Pubmed
Cherny, Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses. 2010, Pubmed
Chou, Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing. 2000, Pubmed
Cibois, A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA. 2010, Pubmed , Xenbase
Clerte, The domains of polypyrimidine tract binding protein have distinct RNA structural preferences. 2009, Pubmed
Conte, Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold. 2000, Pubmed
Cosson, Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding. 2006, Pubmed , Xenbase
Dasgupta, The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins. 2012, Pubmed
Delaunay, The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element. 2004, Pubmed , Xenbase
Dembowski, The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation. 2009, Pubmed
Deo, Recognition of polyadenylate RNA by the poly(A)-binding protein. 1999, Pubmed
Dujardin, CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3. 2010, Pubmed
Edwards, Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1. 2011, Pubmed , Xenbase
Ezzeddine, EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila. 2002, Pubmed , Xenbase
Faustino, Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment. 2005, Pubmed
Garner, Independent interactions of ubiquitin-binding domains in a ubiquitin-mediated ternary complex. 2011, Pubmed
Gautier-Courteille, EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation. 2004, Pubmed , Xenbase
Goo, CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA. 2009, Pubmed
Gooding, Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon. 1998, Pubmed
Graindorge, Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis. 2008, Pubmed , Xenbase
Handa, Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. 1999, Pubmed
Huichalaf, Regulation of the promoter of CUG triplet repeat binding protein, Cugbp1, during myogenesis. 2007, Pubmed
Iakova, Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate. 2004, Pubmed
Izquierdo, Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. 2005, Pubmed
Joshi, Crystallographic analysis of polypyrimidine tract-binding protein-Raver1 interactions involved in regulation of alternative splicing. 2011, Pubmed
Kafasla, Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation. 2009, Pubmed
Kalsotra, A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. 2008, Pubmed
Koshelev, Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1. 2010, Pubmed
Kress, Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice. 2007, Pubmed
Kuyumcu-Martinez, Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. 2007, Pubmed
Lee, Pathogenic mechanisms of myotonic dystrophy. 2009, Pubmed
Legagneux, Substrate-specific regulation of RNA deadenylation in Xenopus embryo and activated egg extracts. 1995, Pubmed , Xenbase
Lin, Regulation of alternative 3' splice site selection by constitutive splicing factors. 1995, Pubmed
Marquis, CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding. 2006, Pubmed , Xenbase
Masuda, CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay. 2012, Pubmed
Meijer, Translational control of maskin mRNA by its 3' untranslated region. 2007, Pubmed , Xenbase
Michalowski, Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. 1999, Pubmed
Miller, Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. 2000, Pubmed
Moraes, CUG-BP binds to RNA substrates and recruits PARN deadenylase. 2006, Pubmed , Xenbase
Mori, Quantitative analysis of CUG-BP1 binding to RNA repeats. 2008, Pubmed
Mukhopadhyay, Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2. 2003, Pubmed
Nakamura, Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. 2004, Pubmed
Oberstrass, Structure of PTB bound to RNA: specific binding and implications for splicing regulation. 2005, Pubmed
Paillard, c-Jun ARE targets mRNA deadenylation by an EDEN-BP (embryo deadenylation element-binding protein)-dependent pathway. 2002, Pubmed , Xenbase
Paillard, A functional deadenylation assay identifies human CUG-BP as a deadenylation factor. 2003, Pubmed , Xenbase
Paillard, EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos. 1998, Pubmed , Xenbase
Pérez, Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo. 1997, Pubmed
Pérez-Cañadillas, Grabbing the message: structural basis of mRNA 3'UTR recognition by Hrp1. 2006, Pubmed
Qin, Monitoring RNA base structure and dynamics using site-directed spin labeling. 2003, Pubmed
Ray, Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. 2009, Pubmed
Salisbury, Ectopic expression of cyclin D3 corrects differentiation of DM1 myoblasts through activation of RNA CUG-binding protein, CUGBP1. 2008, Pubmed
Sharma, U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression. 2011, Pubmed
Sharma, Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. 2008, Pubmed
Simpson, Structure and RNA interactions of the N-terminal RRM domains of PTB. 2004, Pubmed
Singh, Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. 1995, Pubmed
Teplova, Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1. 2008, Pubmed
Teplova, Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1. 2010, Pubmed
Timchenko, CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. 1999, Pubmed
Timchenko, Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis. 2004, Pubmed
Timchenko, Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. 1996, Pubmed
Timchenko, Age-specific CUGBP1-eIF2 complex increases translation of CCAAT/enhancer-binding protein beta in old liver. 2006, Pubmed
Tsuda, Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3. 2009, Pubmed
Vlasova, Posttranscriptional regulation of gene networks by GU-rich elements and CELF proteins. 2008, Pubmed
Vlasova, Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1. 2008, Pubmed
Wachowius, Chemical RNA modifications for studies of RNA structure and dynamics. 2010, Pubmed
Wagner, Polypyrimidine tract binding protein antagonizes exon definition. 2001, Pubmed
Wang, Structural basis for recognition of AU-rich element RNA by the HuD protein. 2001, Pubmed
Ward, CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1. 2010, Pubmed
Xue, Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. 2009, Pubmed
Zhang, The RNA-binding protein CUGBP1 regulates stability of tumor necrosis factor mRNA in muscle cells: implications for myotonic dystrophy. 2008, Pubmed
Zhang, Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. 2008, Pubmed
Zuker, Mfold web server for nucleic acid folding and hybridization prediction. 2003, Pubmed
de Moor, The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes. 1997, Pubmed , Xenbase
de Moor, Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA. 1999, Pubmed , Xenbase