Factors that control translational fidelity in mammalian cells

翻译通读（Translational readthrough，TR）发生在核糖体继续合成蛋白质超过终止密码子并在C末端延伸该蛋白质时，提供新的定位信号或与新伴侣相互作用的可能性。在该基金的前一个任期内，我们发现TR可以在哺乳动物细胞中用适当的双荧光素酶报告基因进行评估。我们发现TR在衰老细胞中降低，并且这种降低依赖于视网膜母细胞瘤（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使衰老细胞中的RB途径失活时，TR活性恢复。RB通过干扰RNA聚合酶III抑制tRNA基因的转录。首先，我们将过表达已知与无义密码子UAA或UAG（tRNATyr、tRNAGln、tRNALys）接近同源的天然tRNA，并且我们预期这种过表达将恢复衰老细胞中的TR效率。其次，为了表征RB途径失活刺激的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，映射哺乳动物细胞中的TR事件，并表征可能受到这种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.