title: Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.
abstract: Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3' major domain of the 20S pre-ribosomal RNA.
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.
authors.forename: Ralph D
authors.initials: RD
authors.lastname: Hector
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK.
authors.forename: Elena
authors.initials: E
authors.lastname: Burlacu
authors.affiliation: MRC Human Genetics Unit, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: Aitken
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.
authors.forename: Thierry
authors.initials: T
authors.lastname: Le Bihan
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.
authors.forename: Maarten
authors.initials: M
authors.lastname: Tuijtel
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.
authors.forename: Alina
authors.initials: A
authors.lastname: Zaplatina
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK.
authors.forename: Atlanta G
authors.initials: AG
authors.lastname: Cook
authors.affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK sgrannem@staffmail.ed.ac.uk.
authors.forename: Sander
authors.initials: S
authors.lastname: Granneman
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D008958:Models, Molecular; D015233:Models, Statistical; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D012322:RNA Precursors / Q000737:chemistry* / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000378:metabolism; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D009711:Nucleotides; D012322:RNA Precursors; D012335:RNA, Ribosomal; C501334:RPS3 protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins