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Quantitative mass spectrometry analysis of 70S ribosomes from iSAT

ABSTRACT: The ribosome is one of the largest and the most complicated enzymes in cells, making up 25% of the bacterial cell’s dry mass, and is responsible for protein synthesis in all organisms on earth. In 2013, Jewett et al. reported an in vitro system named integrated rRNA synthesis, ribosome assembly, and translation (iSAT) reaction, which provided a defined system for ribosome biogenesis in a near-physiological environment.In this project, the r-protein composition in 30S and 50S ribosomeal subunits assembled in the iSAT reaction was quantified by mass spectrometry compared to 15N labeled 70S ribosomes.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Escherichia Coli

SUBMITTER: Xiyu Dong

LAB HEAD: James R. Williamson

PROVIDER: PXD038716 | Pride | 2023-05-10

REPOSITORIES: Pride

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Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Dong Xiyu X Doerfel Lili K LK Sheng Kai K Rabuck-Gibbons Jessica N JN Popova Anna M AM Lyumkis Dmitry D Williamson James R JR

Nucleic acids research 20230401 6

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in ...[more]

PMID: 36864669

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R-proteins in modification deficient ribosomes - dissociated 50S subunits from 70S

Project description:Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an E. coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Ribosomal protein composition of the mature 50S ribosomes and free 50S subunits was analyzed by SILAC based qMS approach. We have found that absence of 23S rRNA modifications around PTC does not affect significantly r-protein content of incompletely assembled 50S or mature 50S ribosomes.

2024-03-15 | PXD047588 | Pride

LC-MS/MS analysis of ribosome UFMylation in vitro

Project description:UFMylation, is a Ubiquitin-like modification occurring on RPL26, a ribosomal subunit, upon ribosome stalling at the ER membrane. This modification is achieved by a pool of enzymes commonly referred to as E1-activating, E2-conjugating and E3-ligase enzymes. The UFM1 E3 ligase comprises of three proteins: UFL1, UFBP1 and CDK5RAP3, referred to as UFM1 Ribosome E3 Ligase (UREL) complex. While UFL1 and UFBP1 are sufficient for E3 ligase activity in vitro, CDK5RAP3 is indispensable for ribosome UFMylation in vivo. In this work, we characterize in vitro UFMylation reaction of purified 60S ribosomes in the presence and absence of CDK5RAP3 using LC-MS/MS. Specifically, we analyse sites of UFMylation, linkage type and quantify monoUFMylation and diUFMylation on RPL26. Interestingly, we find UFMylation to occur on a specific lysine residue, K134, in the presence of UFL1/UFBP1 alone and in complex with CDK5RAP3. In addition, we also provide quantitative information on relative abundances of RPL26 mono- and di-UFMylation under these conditions.

2024-03-22 | PXD046991 | Pride

Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density

Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.

2021-06-22 | PXD026828 | Pride

RiboSeq of BSR cells infected at MOI 3 with Theiler's murine encephalomyelitis virus, compared to mock-infected controls, harvested at 10 hpi. Fragments of lengths 35-65 nt purified.

Project description:BSR cells were infected with the Cardiovirus B archetype, Theiler’s murine encephalomyelitis virus (TMEV), and mutants thereof, and subjected to ribosome profiling to analyse ribosome collision events on the viral genome. Samples were harvested at 10 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 35-65nt long, in order to capture fragments protected by disomes. Fragments were cloned into adapters based on the TruSeq small RNA adapter kit, with an additional seven random nucleotides at 5′-end of the 3′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform.

2021-11-10 | E-MTAB-9437 | biostudies-arrayexpress

RiboSeq of BSR cells infected at MOI 3 with Theiler's murine encephalomyelitis virus, compared to mock-infected controls, harvested at 10 hpi

Project description:BSR cells were infected with the Cardiovirus B archetype, Theiler’s murine encephalomyelitis virus (TMEV), and mutants thereof, and subjected to ribosome profiling to analyse the viral translatome, frameshifting, and ribosome pausing events. Samples were harvested at 10 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 19-35nt long. Fragments were cloned into adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform.

2021-11-10 | E-MTAB-9438 | biostudies-arrayexpress

Ribosome profiling of the genome-reduced bacterium Mycoplasma pneumoniae

Project description:In order to study quantitatively the translation efficiency in the genome-reduced bacterium Mycoplasma pneumoniae, we performed ribosome profiling (ribo-seq) in standard growth conditions. We first tested whether the ribosome profiling method could be applied to M. pneumoniae and assessed the quality of the obtained data. Mpn present some characteristics that differ from other model bacterial species where ribosome profiling has been established, like the absence of a cell wall, different lipid composition and low number of ribosomes (200-300 per cell compared with ~55,000 in E. coli). Thus, several aspects of the original method (Ingolia et al., 2009) had to be adapted. As a reference, we also performed ribosome profiling in E. coli. We also calibrated the ribosome P-site shift from the 3’ end of ribosome protected footprints (RPFs) by analyzing the metagene profile of a sample treated with tetracycline.

2023-07-01 | E-MTAB-11935 | biostudies-arrayexpress

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5'UTR and Responsive to mTORC1 Signaling (RNA-Seq)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5M-bM-^@M-^Y untranslated region (5M-bM-^@M-^Y UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Monitor the translational status of transcriptome in mammalian cells under different conditions

2011-11-12 | E-GEOD-25626 | biostudies-arrayexpress

RNA-seq and TRAP-seq of astrocytes and brain tissue in mouse models of Alzheimer's pathology

Project description:Alzheimer’s disease is known to alter astrocytes, but the effect of Aß and Tau pathology on these cells remains poorly understood. We investigated the transcriptomic behaviour of astrocytes (via translating ribosome affinity purification (TRAP)), and bulk brain tissue, in mouse models of APP/PS1 ß-amyloidopathy and MAPT-P301S tauopathy, in a mouse model overexpressing cytoprotective Nrf2 specifically in astrocytes (GFAP-Nrf2 model), and in crosses between the amyloidopathy and tauopathy models with the GFAP-Nrf2 mouse.

2021-12-02 | E-MTAB-10985 | biostudies-arrayexpress

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions

2012-07-26 | E-GEOD-37744 | biostudies-arrayexpress

Cardiac mitochondrial function depends on BUD23 mediated ribosome programming

Project description:Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells.

2020-01-23 | E-MTAB-8673 | biostudies-arrayexpress

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