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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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE52878 | GEO | 2014/11/14

SECONDARY ACCESSION(S): PRJNA230385

REPOSITORIES: GEO

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

Project 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 3M-bM-^@M-^Y major domain of the 20S pre-ribosomal RNA. Three datasets of yeast ribosomal samples subjected to different chemical modifications; 1M7 dataset contains 8 different modified samples and 2 control samples; NAI dataset contains 3 different modified samples and 2 control samples; DMS dataset contains 1 modified sample and 1 control sample. Each sample consists of at least two replicates.

2014-11-14 | E-GEOD-52878 | biostudies-arrayexpress

PDCD2 functions as an evolutionarily conserved chaperone dedicated for the 40S ribosomal protein uS5

Project description:PDCD2 is an evolutionarily conserved protein that is essential for cell proliferation and embryonic development. To date, however, the function of PDCD2 underlying its fundamental cellular role has remained elusive. Here we used quantitative proteomics approaches to define the protein-protein interaction network of human PDCD2. Our data revealed that PDCD2 specifically interacts with the 40S ribosomal protein uS5 (RPS2) and that the PDCD2-uS5 complex is assembled co-translationally. Loss of PDCD2 expression leas to defects in the synthesis of small ribosomal subunit that phenocopies a uS5 deficiency. Notably, we showed that PDCD2 is important for the accumulation of soluble uS5 proteins as well as its incorporation into 40S ribosomal subunit. Our findings support that the essential molecular function of PDCD2 is to act as a dedicated chaperone that recognizes the ribosomal protein uS5 co-translationally in the cytoplasm and accompanies uS5 to ribosome assembly sites in the nucleolus. As most dedicated ribosomal protein chaperones have been identified in yeast, our study indicates that similar mechanisms exist in human cells to assist ribosomal proteins coordinate their folding, nuclear import, and assembly in pre-ribosomal particles.

2020-09-01 | GSE151094 | GEO

High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast.

Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.

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Proteomics analysis of human pre-40S ribosomal particles

Project description:Proteomics analysis of late human pre-40S particles purified using a catalytically-inactive RIO1 as bait. Analysis of RIO1(kd)-StHA pre-40S particles composition upon RPS26 depletion with siRNA.

2021-05-12 | PXD019270 | Pride

Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) recycle post-termination 40S subunits in vivo

Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.

2018-08-30 | GSE108942 | GEO

Using DMS-MaPseq to dissect structural differences of ribosomal RNA in different pre-40S intermediates

Project description:Ribosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head.

2021-06-04 | GSE160161 | GEO

Using DMS-MaPseq to dissect structural differences of ribosomal RNA in different pre-40S intermediates

Project description:Using DMS-MaPseq to dissect structural differences of ribosomal RNA in different pre-40S intermediates

| PRJNA672155 | ENA

40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

Project description:The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we developed an enhanced 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

2021-03-01 | GSE145904 | GEO

The global map of Rio1 activity in Saccharomyces cerevisiae

Project description:In Saccharomyces cerevisiae, the kinase Rio1 regulates rDNA transcription and segregation, pre-rRNA cleavage, and 40S ribosomal subunit maturation. Other roles are unknown. Human orthologue RIOK1; which is frequently overexpressed in malignancies, drives tumor growth and metastasis. Again, also RIOK1 biology is poorly understood. In this study, we charted the global activity of Rio1 in budding yeast. By producing and systems-integrating its protein-interaction, gene-transcription, and chromatin-binding maps we generated Rio1's multi-layered activity network, which controls protein synthesis and turnover, metabolism, growth, proliferation, and genetic stability. Rio1 regulates itself at the transcriptional level, and manages its network both directly and indirectly, via a battery of regulators and transcription factors, including Gcn4. We experimentally confirmed the network and show that Rio1 commands its downstream circuit depending on the growth conditions encountered. We also find that Rio1 and RIOK1 activities are functionally equivalent. Our data suggest that pathological RIOK1 expression may deregulate its network and fuel promiscuous transcription and ribosome production, uncontrolled metabolism, growth, proliferation, and chromosomal instability; well-known contributors to cancer initiation, maintenance and metastasis.

2020-11-23 | GSE107318 | GEO

AP-MS analysis of human RIOK1 and USP16

Project description:To discover novel factors involved in the final steps of 40S ribosomal subunit biogenesis in human cells, we isolated late cytoplasmic 40S subunit precursors by affinity purification (AP) of the assembly factor RIOK1. Subsequent analysis of their composition by mass spectrometry (MS) led to identification of the deubiquitinase USP16. AP-MS of USP16 confirms that it is a component of late cytoplasmic pre-40S particles and we further show that USP16 possesses a new, ribosome-associated function.

2020-03-04 | PXD016458 | Pride

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