Departamento de Genética, Universidad de Sevilla, E-41012 Sevilla, Spain.
J Biol Chem. 2012 Nov 2;287(45):38390-407. doi: 10.1074/jbc.M112.400564. Epub 2012 Sep 20.
Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.
大多数核糖体蛋白在核糖体的生物发生和功能中发挥重要作用。在这里，我们研究了在酵母酿酒酵母中必需核糖体蛋白 L40 在这些过程中的贡献。RPL40A 或 RPL40B 基因的缺失以及体内 L40 的耗竭会损害 60S 核糖体亚基的生物发生。多核糖体谱分析显示半分子的积累和游离 60S 核糖体亚基的适度减少。在 L40 耗尽的菌株中进行的脉冲追踪、Northern 印迹和引物延伸分析清楚地表明，L40 不是前 rRNA (pre-rRNA) 加工反应所必需的，但有助于最佳 27SB pre-rRNA 成熟。此外，L40 的耗竭会阻碍 pre-60S 核糖体颗粒的核质输出。重要的是，所有这些缺陷很可能是由于细胞质 pre-60S 核糖体亚基中 Nmd3 和 Rlp24 的释放受损以及它们向核（质）体的有效再循环受阻而直接导致的。我们表明，带有血凝素表位标签的 L40A 组装在细胞质中成为几乎成熟的 pre-60S 核糖体颗粒。最后，我们已经确定带有血凝素表位标签的 L40A 赋予了对 sordarin 的抗性，sordarin 是一种翻译抑制剂，会损害真核延伸因子 2 的功能，而 rpl40a 和 rpl40b 缺失突变体对此抗生素敏感。我们得出的结论是，L40 是在 pre-60S 核糖体亚基的非常晚期组装的，并且其掺入 60S 核糖体亚基是亚基连接的前提条件，并且可能确保易位过程的正确功能。
