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Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution

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. T...

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Autores principales: Hector, Ralph D., Burlacu, Elena, Aitken, Stuart, Bihan, Thierry Le, Tuijtel, Maarten, Zaplatina, Alina, Cook, Atlanta G., Granneman, Sander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231735/
https://www.ncbi.nlm.nih.gov/pubmed/25200078
http://dx.doi.org/10.1093/nar/gku815
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author Hector, Ralph D.
Burlacu, Elena
Aitken, Stuart
Bihan, Thierry Le
Tuijtel, Maarten
Zaplatina, Alina
Cook, Atlanta G.
Granneman, Sander
author_facet Hector, Ralph D.
Burlacu, Elena
Aitken, Stuart
Bihan, Thierry Le
Tuijtel, Maarten
Zaplatina, Alina
Cook, Atlanta G.
Granneman, Sander
author_sort Hector, Ralph D.
collection PubMed
description 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.
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spelling pubmed-42317352014-11-21 Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution Hector, Ralph D. Burlacu, Elena Aitken, Stuart Bihan, Thierry Le Tuijtel, Maarten Zaplatina, Alina Cook, Atlanta G. Granneman, Sander Nucleic Acids Res RNA 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. Oxford University Press 2014-10-29 2014-09-08 /pmc/articles/PMC4231735/ /pubmed/25200078 http://dx.doi.org/10.1093/nar/gku815 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA
Hector, Ralph D.
Burlacu, Elena
Aitken, Stuart
Bihan, Thierry Le
Tuijtel, Maarten
Zaplatina, Alina
Cook, Atlanta G.
Granneman, Sander
Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title_full Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title_fullStr Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title_full_unstemmed Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title_short Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution
title_sort snapshots of pre-rrna structural flexibility reveal eukaryotic 40s assembly dynamics at nucleotide resolution
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231735/
https://www.ncbi.nlm.nih.gov/pubmed/25200078
http://dx.doi.org/10.1093/nar/gku815
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