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Gerhalter, M.* ; Kofler, L.* ; Zisser, G.* ; Merl-Pham, J. ; Hauck, S.M. ; Bergler, H.*

The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

RNA 30, 807-823 (2024)
Verlagsversion DOI PMC
Open Access Gold (Paid Option)
Creative Commons Lizenzvertrag
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 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 reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for 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 is lacking 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 for the assembly process.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Aberrant Pre-rrna ; Pre-rrna Processing ; Primer Extension ; Reverse Transcription ; Ribosome Biogenesis; Saccharomyces-cerevisiae; Polynucleotide Kinase; Ribonuclease Mrp; Pin Domain; Final Step; Yeast; Cleavage; Subunit; Protein; 5.8s
ISSN (print) / ISBN 1355-8382
e-ISSN 1469-9001
Zeitschrift RNA
Quellenangaben Band: 30, Heft: 7, Seiten: 807-823 Artikelnummer: , Supplement: ,
Verlag Cold Spring Harbor Laboratory Press
Verlagsort 1 Bungtown Rd, Cold Spring Harbor, Ny 11724 Usa
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) CF Metabolomics & Proteomics (CF-MPC)
Förderungen Austrian Science Foundation (FWF)