title: Tagging of functional ribosomes in living cells by HaloTag® technology.
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.
authors.affiliation: San Raffaele Scientific Institute-DIBIT, via Olgettina 58, 20132, Milan, Italy. gallo.simone@hsr.it
authors.forename: Simone
authors.initials: S
authors.lastname: Gallo
authors.forename: Anne
authors.initials: A
authors.lastname: Beugnet
authors.forename: Stefano
authors.initials: S
authors.lastname: Biffo
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010448:Peptide Initiation Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins; C021019:eIF-6