Factors that control translational fidelity in mammalian cells

当核糖体在停止密码子之外继续合成蛋白质并将该蛋白质延伸到c端，提供新的定位信号或与新伴侣相互作用的可能性时，就会发生翻译读通（TR）。在此资助的前任期内，我们发现可以用适当的双荧光素酶报告基因在哺乳动物细胞中评估TR。我们发现TR在衰老细胞中减少，这种减少取决于视网膜母细胞瘤（RB）肿瘤抑制因子的活性。我们的项目包括确定RB减少TR的机制，并描述依赖于RB的TR事件。当使用癌蛋白E7或激酶CDK4在衰老细胞中灭活RB通路时，TR活性恢复。RB通过干扰RNA聚合酶III抑制tRNAs基因的转录。首先，我们将过表达与无义密码子UAA或UAG接近同源的天然trna (tRNATyr, tRNAGln, tRNALys)，我们期望这种过表达将恢复衰老细胞中的TR效率。其次，为了表征RB通路失活刺激的TR事件，我们将使用核糖体分析和生物信息学。我们的长期目标是提供一种资源来绘制哺乳动物细胞中的TR事件，并表征可能受该机制影响的功能途径。**

Translational readthrough (TR) occurs when ribosomes continue to synthesize a protein beyond a stop codon and extend this protein in the C-terminal, providing a new localization signal or the possibility to interact with a new partner. During the previous tenure of this grant, we found that TR can be assessed in mammalian cells with appropriate dual luciferase reporters. We found that TR is decreased in senescent cells and that this decrease depends on the activity of the retinoblastoma (RB) tumor suppressor. Our project consists in identifying the mechanism by which RB reduces TR and to characterize TR events that depend upon RB. When the RB pathway is inactivated in senescent cells using either the oncoprotein E7 or the kinase CDK4, TR activity is restored. RB represses the transcription of tRNAs genes by interfering with RNA polymerase III. First, we will overexpress natural tRNAs known to be near cognate to non-sense codons UAA or UAG (tRNATyr, tRNAGln, tRNALys) and we expect that this overexpression will restore TR efficiency in senescent cells. Second, to characterize TR events stimulated by RB pathway inactivation, we will use ribosome profiling and bioinformatics. Our long- term goal is to provide a resource that maps TR events in mammalian cells and to characterize the functional pathways that can be potentially affected by this mechanism. **