Tagging of functional ribosomes in living cells by HaloTag® technology

Article
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Abstract

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following of protein synthesis in vivo and isolating translated mRNAs. HaloTag® is a new technology which allows detection in living cells, biochemical purification, and localization studies. In the present work, we tested HaloTag®-based ribosomal tagging. We focused on eIF6 (eukaryotic Initiation Factor 6 free 60S ribosomal marker), RACK1 (Receptor for Activated C Kinase 1; 40S and polysomes, not nuclear), and rpS9 (40S ribosomes, both in the nucleus and in the cytoplasm). Experiments performed on HEK293 cells included ribosomal profiles and Western blot on the fractions, purification of HaloTag® proteins, and fluorescence with time-lapse microscopy. We show that tagged proteins can be incorporated on ribosomes and followed by time-lapse microscopy. eIF6 properly accumulates in the nucleolus, and it is redistributed upon actinomycin D treatment. RACK1 shows a specific cytoplasmic localization, whereas rpS9 is both nucleolar and cytoplasmic. However, efficiency of purification varies due to steric hindrances. In addition, the level of overexpression and degradation may vary upon different constructs. In summary, HaloTag® technology is highly suitable to ribosome tagging, but requires prior characterization for each construct.

Keywords

eIF6 RACK1 rpS9 Polysomal profiles 
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Notes

Acknowledgments

We thank Jacqui Mendez of Promega Corporation who assisted us with indications about Promega products and Cesare Covino of ALEMBIC (Advance Light and Electron Microscopy Bio Imaging Center) who took the images of fluorescence tagged cells. We thank Viviana Volta for reading the manuscript and for technical advice.

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Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  1. 1.San Raffaele Scientific Institute-DIBITMilanItaly
  2. 2.University of Eastern PiedmontAlessandriaItaly

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