| Abstrakt: |
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., 27SA2, 27SA3, 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. |
