title: Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies.
abstract: Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method.
authors.affiliation: Institute of Biochemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Emanuel
authors.initials: E
authors.lastname: Wyler
authors.forename: Mirjam
authors.initials: M
authors.lastname: Zimmermann
authors.forename: Barbara
authors.initials: B
authors.lastname: Widmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Gstaiger
authors.forename: Jens
authors.initials: J
authors.lastname: Pfannstiel
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kutay
authors.forename: Ivo
authors.initials: I
authors.lastname: Zemp
mesh_terms: D015152:Blotting, Northern; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D017346:Protein Serine-Threonine Kinases; C542171:RIOK2 protein, human