Factors that control translational fidelity in mammalian cells

当核糖体继续合成超过终止密码子的蛋白质并在C末端延伸该蛋白质，提供新的定位信号或与新伙伴相互作用的可能性时，就会发生翻译通读(Translational Read Throughing，TR)。在这项资助的前一个任期内，我们发现在哺乳动物细胞中可以用适当的双重荧光素酶报告基因来评估tr。我们发现，在衰老的细胞中，TR减少，并且这种减少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，并表征依赖于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的活性就会恢复。Rb通过干扰RNA聚合酶III抑制tRNAs基因的转录。首先，我们将过度表达已知与无义密码子UAA或UAG(tRNatyr，tRNAGln，tRNALys)同源的天然tRNAs，我们预计这种过度表达将恢复衰老细胞的tR效率。其次，我们将使用核糖体图谱和生物信息学来表征Rb途径失活所刺激的TR事件。我们的长期目标是提供一种在哺乳动物细胞中映射TR事件的资源，并表征可能受这一机制影响的功能通路。**

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. **