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Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors.

ABSTRACT: Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway.

SUBMITTER: Shao S

PROVIDER: S-EPMC4175178 | biostudies-other | 2014 Sep

REPOSITORIES: biostudies-other

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Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors.

Shao Sichen S Hegde Ramanujan S RS

Molecular cell 20140814 6

Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 i ...[more]

PMID: 25132172

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Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.

Dataset Information

Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors.

Publications

Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors.

Similar Datasets

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