Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins
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Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins. / Jovanovic, Bogdan; Schubert, Lisa; Poetz, Fabian; Stoecklin, Georg.
In: Archives of Biological Sciences, Vol. 73, No. 1, 2021, p. 47-55.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins
AU - Jovanovic, Bogdan
AU - Schubert, Lisa
AU - Poetz, Fabian
AU - Stoecklin, Georg
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - affinity purification
KW - ribosome purification
KW - ribosome-associated proteins
KW - RPS9
KW - streptavidin-binding peptide
U2 - 10.2298/ABS20120557J
DO - 10.2298/ABS20120557J
M3 - Journal article
AN - SCOPUS:85103490296
VL - 73
SP - 47
EP - 55
JO - Archives of Biological Sciences
JF - Archives of Biological Sciences
SN - 0354-4664
IS - 1
ER -
ID: 260605768