Suppression of 15-lipoxygenase synthesis by hnRNP E1 is dependent on repetitive nature of LOX mRNA 3'-UTR control element DICE.
Cytidine-rich 15-lipoxygenase differentiation control element (15-LOX DICE) is a multifunctional cis-element found in the 3'-UTR of numerous eukaryotic mRNAs. It binds KH domain proteins of the type hnRNP E and K, thus mediating mRNA stabilization and translational control. Translational silencing is caused by formation of a simple binary complex between DICE and recombinant hnRNP E1 (E1). Electromobility shift assays and sucrose gradient centrifugation demonstrate that rabbit 15-LOX DICE, which is composed of ten subunits of the sequence (CCCCPuCCCUCUUCCCCAAG)10=10R, is able to bind up to ten molecules of E1. Protein/RNA interaction was studied with different subunits and submotifs of the 10R structure. Binding appears to be dependent on the degree of polymerization of the C-clusters (1R<2R<4R<10R), but not on their order. The minimal motif, which still functioned in E1 binding, contained two C-clusters (CCCCPuCCCUCUU). For efficient translational control, E1 binding is a necessary, but not sufficient, condition. Translational inhibition by E1 is only observed when at least a dimeric 2R configuration of the DICE is present in the 3'-UTR of a reporter mRNA. We conclude that binding of at least two E1 molecules activate or expose a binding site to enable the complex to interact with the 5'-end of the mRNA and the translational machinery. DICE-motifs are widely distributed in nature. The UTR database UTRnr contains 78 entries of mRNAs with 15-LOX DICEs. Most DICEs were two- to fourfold repetitive, but also highly repetitive structures were found, as in quail myelin protein mRNA (31 repeats) and hyperglycemic hormone mRNA of two crayfish species (nine and 11 repeats).
Reimann
Iris
I
Biomedizinisches Forschungszentrum (BMFZ) University Clinics Charité, Augustenburger Platz 1, Humboldt-University Berlin, D-13353, Germany.
Huth
Antje
A
Thiele
Holger
H
Thiele
Bernd-Joachim
BJ
D020413:3' Untranslated Regions / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics*; D060829:Arthropod Proteins; D003400:Astacoidea / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004268:DNA-Binding Proteins; D030541:Databases, Genetic; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007447:Invertebrate Hormones / Q000235:genetics; D008969:Molecular Sequence Data; D009186:Myelin Sheath / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D011784:Quail / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D016415:Sequence Alignment; D020080:Tandem Repeat Sequences / Q000235:genetics
D020413:3' Untranslated Regions; D060829:Arthropod Proteins; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; C094604:PCBP1 protein, human; D016601:RNA-Binding Proteins; C015947:hyperglycemic hormone, crustacean; D001093:Arachidonate 15-Lipoxygenase