Abstract
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.
Highlights
The fate of mRNAs has recently emerged as an important point of regulation of gene expression. mRNA localization, stability and protein translation are closely controlled
Some of them are able to shuttle between the nucleus and the cytoplasm: in this manner nuclear and cytoplasmic events of mRNA metabolism are interconnected [2]. mRNP particle assembly is so integral to gene expression control that it is functionally conceptualized as a posttranscriptional "operon" [3, 4]
Cloning of Tino - In order to identify trans-acting factors that interact with the bcl-2 AU-rich elements (ARE), a human placenta cDNA library was screened by the RNA Three-Hybrid System assay (RNA THS)
Summary
The fate of mRNAs has recently emerged as an important point of regulation of gene expression. mRNA localization, stability and protein translation are closely controlled. RESULTS Cloning of Tino - In order to identify trans-acting factors that interact with the bcl-2 ARE, a human placenta cDNA library was screened by the RNA THS.
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