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

Authors

  • Bogdan Jovanovic 1. Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim; 2. Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg; 3. German Cancer Research Center (DKFZ) – ZMBH Alliance, Heidelberg; 4. Center for Human Molecular Genetics, Faculty of Biology, University of Belgrade, Belgrade
  • Lisa Schubert 1. Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 2. Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany; 3. German Cancer Research Center (DKFZ) – ZMBH Alliance, Heidelberg; 4. Novo Nordiks Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
  • Fabian Poetz 1. Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim; 2. Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg; 3. German Cancer Research Center (DKFZ) – ZMBH Alliance, Heidelberg
  • Georg Stoecklin 1. Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim; 2. Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg; 3. German Cancer Research Center (DKFZ) – ZMBH Alliance, Heidelberg

DOI:

https://doi.org/10.2298/ABS20120557J

Keywords:

RPS9, ribosome-associated proteins, ribosome purification, streptavidin-binding peptide, affinity purification

Abstract

Paper description:

  • There are no reliable tools for the purification and identification of ribosomal and ribosome-associated proteins in human cell lines.
  • We developed a system for the purification of ribosomes and ribosome-associated proteins that can be used in any human cell line.
  • All ribosomal proteins and many ribosome-associated proteins in the HeLa cell line were purified and detected, demonstrating the efficiency of the method.
  • 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 exposure of human cells to different stimuli and conditions.

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.

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References

Caetano-Anollés G, Caetano-Anollés D. Computing the origin and evolution of the ribosome from its structure - Uncovering processes of macromolecular accretion benefiting synthetic biology. Comput Struct Biotechnol J. 2015;13:427-47. https://doi.org/10.1016/j.csbj.2015.07.003

Belin S, Hacot S, Daudignon L, Therizols G, Pourpe S, Mertani HC, Rosa-Calatrava M, Diaz J. Purification of ribosomes from human cell lines. Curr Protoc Cell Biol. 2010;Chapter 3:Unit 3.40. https://doi.org/10.1002/0471143030.cb0340s49

Joazeiro CAP. Mechanisms and functions of ribosome-associated protein quality control. Nat Rev Mol Cell Biol. 2019;20(6):368-83. https://doi.org/10.1038/s41580-019-0118-2

Darnell JC, Van Driesche SJ, Zhang C, Hung KYS, Mele A, Fraser CE, Stone EF, Chen C, Fak JJ, Chi SW, Licatalosi DD, Richter JD, Darnell RB. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell. 2011;146(2):247-61. https://doi.org/10.1016/j.cell.2011.06.013

Drozd M, Delhaye S, Maurin T, Castagnola S, Grossi M, Brau F,Jarjat M, WillemsenR, Capovilla M, Hukema RK, Lalli E, Bardoni B. Reduction of Fmr1 mRNA Levels Rescues Pathological Features in Cortical Neurons in a Model of FXTAS. Mol Ther Nucleic Acids. 2019;18:546-53. https://doi.org/10.1016/j.omtn.2019.09.018

Anderson P, Kedersha N, Ivanov P. Stress granules, P-bodies and cancer. Biochim Biophys Acta.. 2015;1849(7):861-70.

Narla A, Ebert BL. Ribosomopathies: human disorders of ribosome dysfunction. Blood. 2010;115(16):3196-205. https://doi.org/10.1182/blood-2009-10-178129

Anczuków O, Krainer AR. Splicing-factor alterations in cancers. RNA. 2016;22(9):1285-301. https://doi.org/10.1261/rna.057919.116

Israelsen WJ, Dayton TL, Davidson SM, Fiske BP, Hosios AM, Bellinger G, Li J, Yu Y, Sasaki M, Horner JW, Burga LN, Xie J, Jurczak MJ, DePinho RA, Clish CB, Jacks T, Kibbey RG, Wulf GM, Di Vizio D, Mills GB, Cantley LC, Vander Heiden MG. PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell. 2013;155(2):397-409. https://doi.org/10.1016/j.cell.2013.09.025

Li D, Wang J. Ribosome heterogeneity in stem cells and development. J Cell Biol. 2020;219(6):e202001108.

Ferretti MB, Karbstein K. Does functional specialization of ribosomes really exist? RNA. 2019;25(5):521-38. https://doi.org/10.1261/rna.069823.118

Xue S, Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol. 2012;13(6):355-69. https://doi.org/10.1038/nrm3359

Singleton RS, Liu-Yi P, Formenti F, Ge W, Sekirnik R, Fischer R, Adam J, Pollard PJ, Wolf A, Thalhammer A, Loenarz C, Flashman E, Yamamoto A, Coleman ML, Kessler BM, Wappner P, Schofield CJ, Ratcliffe PJ, Cockman ME. OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation. Proc Natl Acad Sci USA. 2014;111(11):4031-6. https://doi.org/10.1073/pnas.1314482111

Reschke M, Clohessy JG, Seitzer N, Goldstein DP, Breitkopf SB, Schmolze DB, Ala U, Asara JM, Beck AH, Pandolfi PP. Characterization and Analysis of the Composition and Dynamics of the Mammalian Riboproteome. Cell Reports. 2013;4(6):1276-87. https://doi.org/10.1016/j.celrep.2013.08.014

Simsek D, Tiu GC, Flynn RA, Byeon GW, Leppek K, Xu AF, Chang HY , Barna M. The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell. 2017;169(6):1051-65.e18. https://doi.org/10.1016/j.cell.2017.05.022

Sanz E, Yang L, Su T, Morris DR, McKnight GS, Amieux PS. Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A. 2009;106(33):13939-44. https://doi.org/10.1073/pnas.0907143106

Gallo S, Beugnet A, Biffo S. Tagging of functional ribosomes in living cells by HaloTag® technology. In Vitro Cell Dev Biol Animal. 2011;47(2):132-8. https://doi.org/10.1007/s11626-010-9370-7

Haneke K, Schott J, Lindner D, Hollensen AK, Damgaard CK, Mongis C, Knop M, Palm W, Ruggieri A, Stoecklin G.CDK1 couples proliferation with protein synthesis. J Cell Biol. 2020;219(3):e201906147. https://doi.org/10.1083/jcb.201906147

Hofmann S, Cherkasova V, Bankhead P, Bukau B, Stoecklin G. Translation suppression promotes stress granule formation and cell survival in response to cold shock.Mol Biol Cell. 2012;23(19):3786-800. https://doi.org/10.1091/mbc.e12-04-0296

Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. 2017;18(5):285-98. https://doi.org/10.1038/nrm.2017.7

Jain S, Wheeler JR, Walters RW, Agrawal A, Barsic A, Parker R. ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure. Cell. 2016;164(3):487-98. https://doi.org/10.1016/j.cell.2015.12.038

Mi H, Muruganujan A, Thomas PD. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 2013;41(Database issue):D377-386. https://doi.org/10.1093/nar/gks1118

Thomas PD. PANTHER: A Library of Protein Families and Subfamilies Indexed by Function. Genome Research. 2003;13(9):2129-41. https://doi.org/10.1101/gr.772403

Pomaznoy M, Ha B, Peters B. GOnet: a tool for interactive Gene Ontology analysis. BMC Bioinformatics. 2018;19(1):470. https://doi.org/10.1186/s12859-018-2533-3

Ederth J, Mandava CS, Dasgupta S, Sanyal S. A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli. Nucleic Acids Res. 2009;37(2):e15. https://doi.org/10.1093/nar/gkn992

Inada T, Winstall E, Tarun SZ, Yates JR, Schieltz D, Sachs AB. One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs. RNA. 2002;8(7):948-58. https://doi.org/10.1017/s1355838202026018

Thomas A, Lee P-J, Dalton JE, Nomie KJ, Stoica L, Costa-Mattioli M, Chang P, Nuzhdin S, Arbeitman MN, Dierick HA. A versatile method for cell-specific profiling of translated mRNAs in Drosophila. PLoS One. 2012;7(7):e40276. https://doi.org/10.1371/journal.pone.0040276

Ohn T, Kedersha N, Hickman T, Tisdale S, Anderson P. A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly. Nat Cell Biol. 2008;10(10):1224-31. https://doi.org/10.1038/ncb1783

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Published

2021-03-19

How to Cite

1.
Jovanovic B, Schubert L, Poetz F, Stoecklin G. Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins . Arch Biol Sci [Internet]. 2021Mar.19 [cited 2024Apr.20];73(1):47-55. Available from: https://www.serbiosoc.org.rs/arch/index.php/abs/article/view/6014

Issue

Vol. 73 No. 1 (2021)

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