title: Interaction network of the ribosome assembly machinery from a eukaryotic thermophile.
abstract: Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Jochen
authors.identifier: 0000-0002-7441-7867
authors.initials: J
authors.lastname: Baßler
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Yasar Luqman
authors.initials: YL
authors.lastname: Ahmed
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Kallas
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Kornprobst
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Fabiola R
authors.initials: FR
authors.lastname: Calviño
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Marén
authors.initials: M
authors.lastname: Gnädig
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Thoms
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Gunter
authors.initials: G
authors.lastname: Stier
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Sherif
authors.initials: S
authors.lastname: Ismail
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Satyavati
authors.initials: S
authors.lastname: Kharde
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Nestor
authors.initials: N
authors.lastname: Castillo
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Griesel
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Bastuck
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Bradatsch
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Emma
authors.initials: E
authors.lastname: Thomson
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Flemming
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Irmgard
authors.initials: I
authors.lastname: Sinning
authors.affiliation: Biochemistry Center Heidelberg BZH, University of Heidelberg, Heidelberg, 69120, Germany.
authors.forename: Ed
authors.initials: E
authors.lastname: Hurt
mesh_terms: D002597:Chaetomium / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D012269:Ribosomal Proteins
keywords: Brix proteins; Chaetomium thermophilum; UTP-A; UTP-B; interaction map; ribosome biogenesis