Institute of Molecular Biosciences, University of Graz, Graz 8010, Austria.
Metabolomics and Proteomics Core, Helmholtz Center Munich, Munich 80939, Germany.
RNA. 2024 Jun 17;30(7):807-823. doi: 10.1261/rna.079912.123.
Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA, 27SA, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.
核糖体将 mRNA 翻译成蛋白质，是所有生物体所必需的。在真核生物中，核糖体的两个亚基都是以严格的层次顺序快速组装的，从核仁中开始转录共同的前体核糖体 RNA（pre-rRNA）。这个 pre-rRNA 编码了四个成熟 rRNA 中的三个，它们是通过几个连续的内切核酸酶和外切核酸酶加工步骤形成的。从历史上看， northern blot 被用于分析各种不同的 pre-rRNA 物种，只能进行粗略的长度估计。虽然这一限制可以通过引物延伸来克服，但这两种方法通常都使用放射性同位素，既费时又费钱。在这里，我们提出了“Riboprobing”，这是一种基于连接子连接的工作流程，随后进行逆转录和 PCR，用于容易和快速地检测和表征 pre-rRNA 物种及其 5'和 3'末端。使用标准的分子生物学实验室设备，“Riboprobing”允许可靠地区分 northern blot 无法分辨的 pre-rRNA 物种（例如 27SA、27SA 和 27SB pre-rRNA）。该方法可成功用于总细胞提取物以及纯化的前核糖体的分析，以便直接评估突变基因版本或抑制剂的影响。在方法开发过程中，我们鉴定并表征了一个迄今为止未被描述的由 LiCl 抑制引起的异常 pre-rRNA。这个 pre-rRNA 片段跨越从加工位点 A1 到 E，形成一个缺乏大多数早期连接组装因子的小 RNP。这一发现扩展了我们对细胞如何应对严重的 pre-rRNA 加工缺陷的认识，并证明了 5'ETS（外部转录间隔子）对组装过程的严格要求。
