Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins

• Verfasst von: Jovanovic, Bogdan [VerfasserIn]
• Verfasst von: Schubert, Lisa [VerfasserIn]
• Verfasst von: Poetz, Fabian [VerfasserIn]
• Verfasst von: Stoecklin, Georg [VerfasserIn]
• Titel: Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins
• Verf.angabe: Bogdan Jovanovic, Lisa Schubert, Fabian Poetz, and Georg Stoecklin
• Jahr: 2021
• Umfang: 9 S.
• Fussnoten: Gesehen am 02.06.2021 ; First published: December 30, 2020
• Titel Quelle: Enthalten in: Archives of biological sciences
• Ort Quelle: Belgrade : Srpsko Biološko Društvo, 2002
• Jahr Quelle: 2021
• Band/Heft Quelle: 73(2021), 1, Seite 47-55
• ISSN Quelle: 1821-4339
• DOI: doi:10.2298/ABS20120557J
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: affinity purification
• Sach-SW: evolution
• Sach-SW: panther
• Sach-SW: reveals
• Sach-SW: ribosome purification
• Sach-SW: ribosome-associated proteins
• Sach-SW: rps9
• Sach-SW: streptavidin-binding peptide
• Sach-SW: stress granule
• K10plus-PPN: 1759442445

Abstract

Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome-associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.