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Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

ABSTRACT: Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4-NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4-NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.

SUBMITTER: Absmeier E

PROVIDER: S-EPMC7615087 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

Absmeier Eva E Chandrasekaran Viswanathan V O'Reilly Francis J FJ Stowell James A W JAW Rappsilber Juri J Passmore Lori A LA

Nature structural & molecular biology 20230831 9

Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4-NOT inserts a helical bund ...[more]

PMID: 37653243

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Project description:3x 12 mL of translation reaction (6x 2 mL) were run. Each reaction contained 1x cT2-tRNA lysate, 3 ng/µL in vitro transcribed, polyA-tailed rare mRNA, 10 mM Potassium acetate, 500 nM CCR4-NOT and 1 µM CNOT4. Addition of the proteins added ca. another 40 mM NaCl. The reaction was started by the addition of 40.8 µM L-Methionine. The reactions were incubated for 30 min at 32 ºC and kept on ice for 10min before loading 2 ml of translation reaction on 10 mL 15% sucrose in 1x RNC buffer each (50 mM HEPES-KOH pH 7.4, 100 mM Potassium acetate, 5 mM Magnesium acetate). Ribosomes were pelleted for five hours at 38000 rpm and 4 ºC in a SW 40 Ti swinging bucket rotor (max acceleration, deceleration 9). After centrifugation the sucrose was removed, and the pellet was resuspended in 150 µL 1x RNC buffer on ice. For the first 12mL, 4 mL of the reaction were crosslinked with 1 mM, 2 mM or 3 mM BS3, respectively for 30 min on ice. For the second and third 12 mL, 4 mL of the reaction were crosslinked with 0.5 mM, 1 mM or 2 mM BS3, respectively, for 30 min on ice. The crosslinking reactions were quenched by the addition of 5 mM Ammonium bicarbonate. The different crosslinking concentrations of the 12mL were pooled (900 µL total) and incubated on 800 µL 50% modified Cytivia Strep Tactin Sepharose High Performance resin. The resin was treated as previously published (45), to avoid Streptactin cleavage upon on bead digestion of the sample. The sample was incubated for 45 min on ice, while the beads were agitated every 5 min to keep them in suspension. The beads were washed five times with 500 µL 1x RNC buffer. The beads were subsequently kept on ice over-night. The next day the beads were re-suspended in 200 µL denaturation buffer (8M Urea in 100 mM Ammonium bicarbonate (ABC)). The following steps, including trypsin digestions, were performed under constant agitation of 14000 rpm in a heating block. Reduction buffer (200 mM Dithiothreitol (DTT) in 50 mM ABC) was added to a final concentration of 10 mM DTT and incubated for 15 min at 25 ºC. Subsequently, alkylation buffer (500 mM Iodoacetamide (IAA) in 50 mM ABC) was added to a final concentration of 40 mM IAA and incubated for 30 min at 25 ºC in the dark. 10 mM DTT was added to quench residual IAA and 0.5 µg LysC protease (MS grade, Pierce) was added for 4 hours at 25 ºC. The sample was diluted with digestion buffer (100 mM ABC) to ensure a Urea concentration below 1.5 M. 4 µg trypsin (MS grade, Pierce) was added to the sample and incubated for 16 hours at 16 ºC. The digestion was stopped by the addition of 10% Trifluoroacetic acid (TFA) to pH 2.5. Peptides were cleaned-up via stage-tipping (46) and vacuum concentrator dried peptides were stored at -80 ºC until further use.

Dataset Information

Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

Publications

Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

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