Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA
J Cell Biol. 2018 Jul 2;217(7):2503-2518. doi: 10.1083/jcb.201711037. Epub 2018 Apr 24.
Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C site of 27SB pre-rRNA. C cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C cleavage. Interestingly, when C cleavage is directly blocked by depleting or inactivating the C endonuclease, assembly progresses through all other subsequent steps.
核糖体生物发生涉及许多核糖体 RNA（rRNA）前体处理事件，以去除内部和外部转录间隔序列，最终产生三个成熟的 rRNA。27SB 前 rRNA 的 C 位点的内切核酸酶切割是大亚基前 rRNA 加工的最后一步，这需要内部转录间隔区 2 间隔 RNA 的去除。C 切割需要 11 种核糖体蛋白和 14 种核糖体组装因子的层次募集。然而，这些蛋白质在 C 切割中的功能仍然不清楚。在这项研究中，我们对 C 切割所需的蛋白质缺失的影响进行了详细分析，并使用组装 60S 亚基的低温电子显微镜结构解释了这些结果。这项工作表明，这些蛋白质对于包括 60S 亚基两个主要功能中心在内的几个区域的重排是必需的，这表明这些重排事件形成了一个导致 C 切割的检查点。有趣的是，当 C 切割直接被缺失或失活 C 内切核酸酶阻断时，组装会通过所有其他后续步骤进行。
