Two RNA-binding Domains Determine the RNA-binding Specificity of Nucleolin

Serin, Guillaume; Joseph, Gérard; Ghisolfi, Laurence; Bauzan, Marielle; Erard, Marie; Amalríc, François; Bouvet, Philippe

doi:10.1074/jbc.272.20.13109

Cited by 91 publications

(130 citation statements)

References 65 publications

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“…Production of Recombinant Proteins-The different nucleolin mutants were produced as described previously (25). Nucleolin mutants were generated by PCR using Vent TM DNA polymerase (New England Biolabs) and hamster nucleolin cDNA as template.…”

Section: Methodsmentioning

confidence: 99%

“…All three RBDs of U2AF 65 are required for high affinity binding to the polypyrimidine tract (4). The first two RBDs of poly(A)-binding protein and nucleolin bind specifically to polyadenylate RNA (21)(22)(23)) and a short stem-loop structure (24,25), respectively, as efficiently as full-length protein; the function of the last two RBD is not known.…”

mentioning

confidence: 99%

“…Although the first two RBD of nucleolin are required for the specific interaction with the NRE motif (24,25), it was of interest to determine which domains are involved in the binding of the ECM. In this work we show that all four RBD of nucleolin are required for interaction with the ECM.…”

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confidence: 99%

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Two Different Combinations of RNA-binding Domains Determine the RNA Binding Specificity of Nucleolin

Ginisty¹,

Amalríc²,

Bouvet³

2001

Journal of Biological Chemistry

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Nucleolin is an abundant nucleolar protein involved in several steps of ribosome biogenesis. The protein is highly conserved through evolution and possesses four RNA-binding domains (RBD), which are likely to determine its RNA binding specificity. Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal RNA. The second is a short singlestranded RNA sequence, the evolutionary conserved motif (ECM), located five nucleotides downstream of the first processing site in the pre-ribosomal RNA 5 external transcribed spacer. Biochemical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nucleolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed that all four RBDs of hamster nucleolin were required for the interaction with the ECM sequence. This RNA binding specificity is conserved between hamster and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBDs for its interaction with an RNA target, demonstrating that a single protein can use different combinations of RBD to interact specifically with several RNA sequences.Specific RNA-protein interactions play an important role in gene expression. One of the most common protein sequence motifs involved in these interactions is the RNA-binding domain (RBD), 1 also called the RNA recognition motif (RRM) (1, 2). A single or multiple RBD, often in combination with other domains involved in protein-protein interaction, are found in proteins that have diverse functions such as in pre-mRNA maturation and splicing (3, 4), hnRNA packaging (5), and mRNA stability and translation (6, 7).The RBD domain is characterized by two highly conserved motifs called RNP-1 and RNP-2 found within a weakly conserved 80-amino acid sequence (1, 2). The crystal and solution structures of several RBDs have revealed a common structural organization composed of four antiparallel ␤-strands packed against two ␣-helices (Refs. 8 -10; 14). The RNP-1 octapeptide and RNP-2 hexapeptide are located in the ␤1 and ␤3 strands, respectively. Structures of RBD found in tandem show that the two independently folded domains do not interact with each other in Sex-lethal (11, 12), HuC (13), and nucleolin proteins (14). In contrast, in hnRNPA1, the two domains interact extensively (15, 16). Despite the highly conserved structure of the RBD, they show a remarkable ability to interact with diverse RNA sequences and structures. The tertiary structures of several RBD⅐RNA complexes are now available. These are the single RBDs from the U1A and U2BЈЈ protein bound to a stem-loop structure (8,17,18), the two RBDs of Sex-lethal, hnRNPA1, and poly(A)-binding protein bound to a singlestranded RNA (12,16,19), and the first two...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Two Different Combinations of RNA-binding Domains Determine the RNA Binding Specificity of Nucleolin

Ginisty¹,

Amalríc²,

Bouvet³

2001

Journal of Biological Chemistry

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“…6A). The rationale for this change in residue resides in the fact that a Phe to Leu single amino acid mutation has been previously described to abolish the functionality of the RNP motif in the case of the nucleolin protein (29). Each mutant was expressed in E. coli (Fig.…”

Section: Binding Of Tdp-43 Derivatives Lacking the Rrm1 Or Rrm2 Motifmentioning

confidence: 99%

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

Buratti

Baralle

2001

Journal of Biological Chemistry

615

606

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Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.We have recently reported the identification of TDP-43 as a splicing regulator that specifically binds the (UG)m-repeated polymorphic region near the 3Ј-splice site of CFTR exon 9 and down-regulates its recognition by the splicing machinery (1). This region, acting in concert with the adjacent (u)n element, is one of the key cis-acting sequences which regulate the proportion of exon 9 skipping in the mature CFTR mRNA transcript (1-3). Considering that exon 9 skipping produces a non-functional CFTR protein (4, 5) the study of the RNA binding properties of TDP-43 is of considerable importance to gain further insight concerning the potential disease-causing consequences of its binding in vivo. Indeed, the clinical relevance of these studies is highlighted by the existence of a clear association between certain (TG)m(T)n alleles with distinct forms of Cystic Fibrosis (1, 6 -9).In addition, the study of (UG)m elements can provide further insight concerning the mRNA splicing process in general because (UG)m sequences have been described to act as splicing regulatory sequences in different genomic contexts. In fact, in addition to the CFTR gene, the presence of simple (UG)mrepeated sequences has been described to influence the splicing process of at least two other genes: the apolipoprotein AII gene (10) and the human cardiac Na ϩ /Ca 2ϩ exchanger (11). In the Apo AII gene the UG tract was shown to be functionally equivalent to a polypyrimidine tract and required for efficient splicing of Apo AII exon 2 (10) while in the human cardiac Na ϩ /Ca 2ϩ exchanger (11) it acts as a strong intronic splicing enhancer situated in intron 2. It should be noted that in contrast with these two genes, the CFTR (UG)m element was found to possess a strong inhibitory effect on CFTR exon 9 splicing, a property that may probably be linked to its peculiar evolutionary history. In fact, sequencing of the mouse CFTR exon 9 genomic region has shown that in the flanking introns, the (TG)m(T)n regulatory elements are absent and that the intron themselves are of very different length when compared with the human introns (...

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“…The central region of nucleolin protein contains 4 tandem RNA‐binding domains, of which the first 2 determine the RNA‐binding specificity and affinity of nucleolin recognition element ((U/G)CCCG(A/G)). Nucleolin regulates the mRNA stability of target genes and consequently mediates their post‐transcriptional control through binding the nucleolin recognition element to corresponding binding elements of different target genes 17, 18, 19. It has been reported that nucleolin can mediate the expressions of angiogenesis‐related genes, including vascular endothelial growth factor‐A (VEGF‐A) and matrix metalloproteinase‐9 (MMP‐9) 14, 20, 21.…”

Section: Introductionmentioning

confidence: 99%

Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9

Zou

Wang

Liu

et al. 2018

J Cellular Molecular Medi

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Hydroxysafflor Yellow A (HSYA), a most representative ingredient of Carthamus tinctorius L., had long been used in treating ischaemic cardiovascular diseases in China and exhibited prominently anticoagulant and pro‐angiogenic activities, but the underlying mechanisms remained largely unknown. This study aimed to further elucidate the pro‐angiogenic effect and mechanism of HSYA on ischaemic cardiac dysfunction. A C57 mouse model of acute myocardial infarction (AMI) was firstly established, and 25 mg/kg HSYA was intraperitoneally injected immediately after operation and given once, respectively, each morning and evening for 2 weeks. It was found that HSYA significantly improved ischaemia‐induced cardiac haemodynamics, enhanced the survival rate, alleviated the myocardial injury and increased the expressions of CD31, vascular endothelial growth factor‐A (VEGF‐A) and nucleolin in the ischaemic myocardium. In addition, HSYA promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs), enhanced the expressions of nucleolin, VEGF‐A and matrix metalloproteinase‐9 (MMP‐9) in a dose‐ and time‐dependent manner. However, down‐regulation of nucleolin expression sharply abrogated the effect mentioned above of HSYA. Further protein‐RNA coimmunoprecipitation and immunoprecipitation‐RT‐PCR assay showed that nucleolin binded to VEGF‐A and MMP‐9 mRNA and overexpression of nucleolin up‐regulated the mRNA expressions of VEGF‐A and MMP‐9 in the HUVECs through enhancing the stability of VEGF‐A and MMP‐9 mRNA. Furthermore, HSYA increased the mRNA expressions of VEGF‐A and MMP‐9 in the extract of antinucleolin antibody‐precipitated protein from the heart of AMI mice. Our data revealed that nucleolin mediated the pro‐angiogenic effect of HSYA through post‐transcriptional regulation of VEGF‐A and MMP‐9 expression, which contributed to the protective effect of HSYA on ischaemic cardiac dysfunction.

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Two RNA-binding Domains Determine the RNA-binding Specificity of Nucleolin

Cited by 91 publications

References 65 publications

Two Different Combinations of RNA-binding Domains Determine the RNA Binding Specificity of Nucleolin

Two Different Combinations of RNA-binding Domains Determine the RNA Binding Specificity of Nucleolin

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9

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