Donnini M, Lapucci A, Papucci L, Witort E, Jacquier A, Brewer G, Nicolin A, Capaccioli S, Schiavone N

Department of Experimental Pathology and Oncology, School of Medicine, University of Florence, 50134 Florence, Italy.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

Mesh Terms:
3' Untranslated Regions, 5' Untranslated Regions, Amino Acid Motifs, Amino Acid Sequence, Animals, Apoptosis, Base Sequence, Blotting, Northern, Blotting, Western, Caenorhabditis elegans, Cell Line, Conserved Sequence, DNA, Complementary, Databases as Topic, Down-Regulation, Evolution, Molecular, Gene Expression Regulation, Genes, bcl-2, HeLa Cells, Heterogeneous-Nuclear Ribonucleoprotein D, Heterogeneous-Nuclear Ribonucleoprotein K, Humans, Microscopy, Fluorescence, Models, Genetic, Molecular Sequence Data, Plasmids, Protein Binding, Protein Biosynthesis, Protein Structure, Tertiary, RNA Processing, Post-Transcriptional, RNA, Messenger, RNA-Binding Proteins, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleases, Sequence Homology, Amino Acid, Time Factors, Transfection, Two-Hybrid System Techniques, Urochordata

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