Mechanism ofmRNA stability control mediated by AU-rich element: Characterization ofcis-elements andtrans-factors
Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells.
Xu, Nianhua, "Mechanism ofmRNA stability control mediated by AU-rich element: Characterization ofcis-elements andtrans-factors" (1999). Texas Medical Center Dissertations (via ProQuest). AAI9942101.