Protein Translation Activity: A New Measure of Host Immune Cell Activation

Seedhom, Mina O.; Hickman, Heather D.; Wei, Jiajie; David, Alexandre; Yewdell, Jonathan W.

doi:10.4049/jimmunol.1600088

Cited by 27 publications

(32 citation statements)

References 23 publications

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“…A combination of puro labeling and ribosome run-off to measure the translation elongation rate By using puro as a nascent protein tag and anti-puro monoclonal antibodies for its detection, we have previously introduced puro-immunomonitoring and, in particular, flow cytometry to measure translation in living cells and organisms (Schmidt et al, 2009;Seedhom et al, 2016). Surface sensing of translation (SUnSET) allows for measuring global translation in individual cells found in mixed populations grown in vitro or ex vivo.…”

Section: Resultsmentioning

confidence: 99%

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

Argüello

Reverendo

Mendes

et al. 2018

Journal of Cell Science

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The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction.This article has an associated First Person interview with the first author of the paper.

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Section: Resultsmentioning

confidence: 99%

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

Argüello

Reverendo

Mendes

et al. 2018

Journal of Cell Science

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“…We first monitored ribosome activity in the immature cell populations of the BM using the RiboPuromycylation Method (RPM) (David et al 2012;Seedhom et al 2016). This method is based on puromycin incorporation into the A site of elongating ribosome and specific and covalent association with nascent peptidic chains.…”

Section: Hscs Display Low Translational Activitymentioning

confidence: 99%

“…Cells were then rinsed with PBS and incubated in staining buffer (0.05% saponin, 10 mM Glycine, 5% FCS, 1× PBS final) for 15 min. Puromycin detection was then performed by staining with Alexa647-conjugated anti-puromycin antibody in staining buffer for 1 h (see Supplemental Table S1 and Seedhom et al 2016). Finally, cells were rinsed in PBS with bovine serum albumin (BSA) and kept at 4°C until analysis.…”

Section: Ribopuromycylationmentioning

confidence: 99%

Mouse adult hematopoietic stem cells actively synthesize ribosomal RNA

Jarzebowski

Bouteiller

Coqueran

et al. 2018

RNA

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The contribution of basal cellular processes to the regulation of tissue homeostasis has just started to be appreciated. However, our knowledge of the modulation of ribosome biogenesis activity in situ within specific lineages remains very limited. This is largely due to the lack of assays that enable quantitation of ribosome biogenesis in small numbers of cells in vivo. We used a technique, named Flow-FISH, combining cell surface antibody staining and flow cytometry with intracellular ribosomal RNA (rRNA) FISH, to measure the levels of pre-rRNAs of hematopoietic cells in vivo. Here, we show that Flow-FISH reports and quantifies ribosome biogenesis activity in hematopoietic cell populations, thereby providing original data on this fundamental process notably in rare populations such as hematopoietic stem and progenitor cells. We unravel variations in pre-rRNA levels between different hematopoietic progenitor compartments and during erythroid differentiation. In particular, our data indicate that, contrary to what may be anticipated from their quiescent state, hematopoietic stem cells have significant ribosome biogenesis activity. Moreover, variations in pre-rRNA levels do not correlate with proliferation rates, suggesting that cell type-specific mechanisms might regulate ribosome biogenesis in hematopoietic stem cells and progenitors. Our study contributes to a better understanding of the cellular physiology of the hematopoietic system in vivo in unperturbed situations.

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“…To minimize the amount of AHA-tagged proteins already released from ribosomes and achieve optimal on-ribosome polypeptide stabilization, we tested different times of AHA incubation and compared the effect of two small molecules (namely cycloheximide (CHX) and sBlock, an anisomycin-based reagent), which are known to inhibit the activity eukaryotic ribosome, while keeping polypeptides bound to the translating ribosomes (Garreau de Loubresse et al, 2014; Grollman, 1967; Seedhom et al, 2016)…”

Section: Resultsmentioning

confidence: 99%

One-shot analysis of translated mammalian lncRNAs with AHARIBO

Minati¹,

Firrito²,

Piano³

et al. 2020

Preprint

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SUMMARYA vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs.Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized polypeptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs.

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Protein Translation Activity: A New Measure of Host Immune Cell Activation

Cited by 27 publications

References 23 publications

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

Mouse adult hematopoietic stem cells actively synthesize ribosomal RNA

One-shot analysis of translated mammalian lncRNAs with AHARIBO

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