title: Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.
abstract: Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Beril
authors.initials: B
authors.lastname: Tutuncuoglu
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Kaige
authors.initials: K
authors.lastname: Yan
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Hailey
authors.initials: H
authors.lastname: Brown
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yixiao
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.|National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Tan
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Gamalinda
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Zhifei
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Jelena
authors.initials: J
authors.lastname: Jakovljevic
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Chengying
authors.initials: C
authors.lastname: Ma
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Lei
authors.affiliation: Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.|National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: John L
authors.initials: JL
authors.lastname: Woolford
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
mesh_terms: D021581:Active Transport, Cell Nucleus; D001483:Base Sequence; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D003593:Cytoplasm / Q000378:metabolism; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D012399:Rotation; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D021903:DNA, Ribosomal Spacer; C475027:NOG1 protein, S cerevisiae; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C477781:NOG2 protein, S cerevisiae