CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding.
CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.
Marquis
Julien
J
Généthon, CNRS UMR 8115, 1 bis rue de l'Internationale, 91002 Evry cedex 2, France.
Paillard
Luc
L
Audic
Yann
Y
Cosson
Bertrand
B
Danos
Olivier
O
Le Bec
Christine
C
Osborne
H Beverley
HB
D020413:3' Untranslated Regions; D000818:Animals; D052157:Aptamers, Nucleotide / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D015374:Biosensing Techniques; D000067879:CELF1 Protein; D005260:Female; D006801:Humans; D007700:Kinetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D018911:Trinucleotide Repeats; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism*
D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; D000067879:CELF1 Protein; C102843:CELF1 protein, human; C110462:EDEN-specific RNA-binding protein, Xenopus; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029867:Xenopus Proteins