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A Comprehensive Roadmap of the Mammalian Ribo-interactome Connects Metabolism Enzymes with Specialized ER-Ribosomes

ABSTRACT: The ribosome has considerably increased in size during metazoan evolution in the form of an RNA shell that could serve as a platform for yet unknown protein interactions. Here, we have comprehensively identified the mammalian ‘ribo-interactome’ by establishing a ribosome affinity purification method. Our findings reveal a multitude of novel ribosome interacting factors, encompassing unanticipated functional categories including energy metabolism, cell redox homeostasis, as well as key protein and RNA modifying enzymes. These findings led us to characterize ufmylation, a novel posttranslational modification on ribosomes, and define its substrates. We further show that pyruvate kinase, a key enzyme for stem cell and cancer metabolism, is an RNA binding protein that unexpectedly interacts with specialized sub-pools of ribosomes at the endoplasmic reticulum (ER) and coordinates the localization and translation of ER destined mRNAs. Collectively, these studies uncover that the ribo-interactome imbues a new layer of regulatory potential in translating the genome.

ORGANISM(S): Mus musculus

PROVIDER: GSE96998 | GEO | 2017/06/01

SECONDARY ACCESSION(S): PRJNA380363

REPOSITORIES: GEO

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Ribosomal protein RPL26 is the principal target of UFMylation

Project description:UFM1 is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to ER stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and deUFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

2018-12-11 | GSE123539 | GEO

Mechanism of 60S ribosomal subunit recognition and UFMylation by the UFM1 E3 ligase complex

Project description:During co-translational translocation at the endoplasmic reticulum (ER), ribosomes can stall and become covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit RPL26 (uL24). This process, known as UFMylation, is mediated by the UFM1 Ribosome E3 Ligase (UREL) complex, comprised of UFL1, UFBP1, and CDK5RAP3. However, the functional consequences of UFMylation and catalytic mechanisms of UREL are unknown. Here, we present crosslinking-mass spectrometry (XL-MS) data of UREL bound to 60S ribosomes.

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SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum

Project description:Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating Translocation-Associated Quality Control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon, and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.

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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.

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A spectral analysis approach to detects actively translated open reading frames in high resolution ribosome profiling data

Project description:RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions from Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of the three-nucleotide periodicity of Ribo-seq reads via spectral analysis methods. Examination of RNA fragments protected by ribosome

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Ribo-Seq transcriptomic analysis of human RD cells infected with enterovirus strains EV7, EV71, and PV1.

Project description:Ribosome profiling (Ribo-Seq) (maps positions of translating ribosomes on the transcriptome) analysis of human (RD) cells infected with enterovirus strains EV7, EV71, and PV1.

2018-10-19 | E-MTAB-6180 | biostudies-arrayexpress

Mus musculus

Project description:A Comprehensive Roadmap of the Mammalian Ribo-interactome Connects Metabolism Enzymes with Specialized ER-Ribosomes

| PRJNA380363 | ENA

Assembly and functionality of the ribosome with tethered subunits

Project description:Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171), RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.

2019-02-25 | GSE119454 | GEO

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