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AP-MS analysis of human RIOK1 and USP16

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ludovic Gillet

LAB HEAD: Ulrike Kutay

PROVIDER: PXD016458 | Pride | 2020-03-04

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	SAINT_19973.output_IP1.tsv	Tabular
	SAINT_21086.output_IP2.tsv	Tabular
	SampleAnnotation_IP1.txt	Txt
	SampleAnnotation_IP2.txt	Txt
	SpecCountNorm_IP1.tsv	Tabular

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Publications

USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.

Montellese Christian C van den Heuvel Jasmin J Ashiono Caroline C Dörner Kerstin K Melnik André A Jonas Stefanie S Zemp Ivo I Picotti Paola P Gillet Ludovic C LC Kutay Ulrike U

eLife 20200304

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 ...[more]

PMID: 32129764

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The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

Project description:In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes.

2024-01-24 | GSE246163 | GEO

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

MS-based identification of AcP10 interaction partners.

Project description:In times of cellular stress, such as during virus infections, the integrated stress response (ISR) blocks translation initiation through phosphorylation of the essential translation initiation factor eIF2. Phosphorylated eIF2 (p-eIF2) sequesters the eIF2-specific guanidine exchange factor (GEF) eIF2B, thereby preventing eIF2 recycling. Here we describe the first example of a viral ISR antagonist that inhibits the ISR at its most central step: the interplay between p-eIF2 and eIF2B. Using AP-MS, we determine that BW10 binds eIF2B. There, it selectively displaces eIF2B’s inhibitor p-eIF2 without affecting the association of its substrate eIF2. By this mechanism, BW10 renders cellular translation immune to regulation by eIF2 phosphorylation. Thus, under stress conditions BW10 creates the unprecedented situation of high levels of p-eIF2 coinciding with unimpaired translation.

2020-07-24 | PXD011879 | Pride

A novel essential RNA-binding protein complex in Trypanosoma brucei

Project description:ZC3H5 is an essential cytoplasmic trypanosome protein with a single Cx7Cx5Cx3H zinc finger domain. We here show that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. ZC3H5 interacts directly with Tb927.11.4900, which in turn interacts with Tb927.7.3040. Tb927.11.4900 has a circularly permuted GTPase domain, which is required for the Tb927.7.3040 interaction. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which were enriched in the motif (U/A)UAG(U/A). Tethering of ZC3H5, or other complex components, to a reporter repressed its expression. However, depletion of ZC3H5 in vivo did not increase the abundance of ZC3H5-bound mRNAs; instead, counter-intuitively, there were very minor decreases in a few targets. This was accompanied by marked increases in the abundance of very stable mRNAs encoding ribosomal proteins. Depletion also resulted in an increase in monosomes at the expense of large polysomes, and appearance of a "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex is implicated in quality control during the translation of sub-optimal open reading frames, and that its assembly is regulated by GTP hydrolysis and GTP-GDP exchange.

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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

ZNF692 organizes a hub for ribosome maturation enhancing translation in rapidly proliferating cells

Project description:Human cancer cell lines (DLD1 wt or ZNF692 KO, and for IP-proteomics HCT116 transfected with GFP, GFP-ZNF692 and deltaNolsZNF692). 788570, HCT116 transfected with GFP; 788571, HCT116 transfected with GFP-ZNF692; 788572, HCT116 transfected with deltaNolsZNF692; 937612, control 40S subunit from DLD1 cells; 937613, control 60S subunit from DLD1 cells; 937614, control 80S monosome fraction from DLD1 cells; 937615, KO ZNF692 40S subunit from DLD1 cells; 937616, KO ZNF692 60S subunit from DLD1 cells; 937617, KO ZNF692 80S monosome fraction from DLD1 cells; 943031, control 40S subunit from DLD1 cells; 943032, control 60S subunit from DLD1 cells; 943033, control 80S monosome fraction from DLD1 cells; 943034, KO ZNF692 40S subunit from DLD1 cells; 943035, KO ZNF692 60S subunit from DLD1 cells; 943036, KO ZNF692 80S monosome fraction from DLD1 cells

2022-04-18 | MSV000089271 | MassIVE

Hcr1/eIF3j is a 60S ribosomal subunit recycling accessory factor in vivo

Project description:Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examined this ribosome recycling role in vivo by ribosome profiling and reporter assays and found that loss of Hcr1 led to reinitiation of translation in 3’UTRs, consistent with a defect in recycling. However, the defect appeared to be in recycling of the 60S subunit, rather than the 40S subunit, because reinitiation did not require an AUG codon and was suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 could not compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggered higher expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.

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FMR1 targets distinct mRNA sequence elements to regulate protein expression [Affymetrix]

Project description:Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which encodes for the RNA-binding protein (RBP), FMR1 (or FMRP). We report the discovery of the RNA recognition elements (RREs), binding sites, and mRNA targets for wild-type and I304N mutant FMRP isoforms as well as its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP can affect their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/- mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide an invaluable molecular guide towards the pursuit of novel therapeutic targets for these devastating neurological disorders. The mRNA profile of RNA recovered from FLAG-antibody immunoprecipitated FMRP was compared to the mRNA profile of the starting lysate material.

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