title: Atomic modeling of the ITS2 ribosome assembly subcomplex from cryo-EM together with mass spectrometry-identified protein-protein crosslinks.
abstract: The assembly of ribosomal subunits starts in the nucleus, initiated by co-transcriptional folding of nascent ribosomal RNA (rRNA) transcripts and binding of ribosomal proteins and assembly factors. The internal transcribed spacer 2 (ITS2) is a precursor sequence to be processed from the intermediate 27S rRNA in the nucleoplasm; its removal is required for nuclear export of pre-60S particles. The proper processing of the ITS2 depends on multiple associated assembly factors and RNases. However, none of the structures of the known ITS2-binding factors is available. Here, we describe the modeling of the ITS2 subcomplex, including five assembly factors Cic1, Nop7, Nop15, Nop53, and Rlp7, using a combination of cryo-electron microscopy and cross-linking of proteins coupled with mass spectrometry approaches. The resulting atomic models provide structural insights into their function in ribosome assembly, and establish a framework for further dissection of their molecular roles in ITS2 processing.
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, People's Republic of China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wu
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, People's Republic of China.
authors.forename: Dan
authors.initials: D
authors.lastname: Tan
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, 15213.
authors.forename: John L
authors.initials: JL
authors.lastname: Woolford
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, People's Republic of China.
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
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, People's Republic of China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D020285:Cryoelectron Microscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C437314:Cic1 protein, S cerevisiae; C451799:NOP7 protein, S cerevisiae; C568702:Nop15 protein, S cerevisiae; C504135:Nop53 protein, S cerevisiae; D009687:Nuclear Proteins; C084787:RLP7 protein, S cerevisiae; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: CXMS; Cic1; ITS2; Nop15; Nop53; Nop7; Rlp7; cross-linking; cryo-EM; ribosome assembly