Dataset Information

The dissociation of RQT complex on ribosome

ABSTRACT: Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by targeting of the faulty nascent peptides for degradation. However, endogenous RQC inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of colliding di- and preferentially tri-ribosomes. Their distinct architecture is visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1/Rqt2 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in dealing with endogenous substrates to maintain cellular protein homeostasis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Akinori Endo

LAB HEAD: Toshifumi Inada

PROVIDER: PXD017196 | Pride | 2020-02-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190927_Ribosomes_1-1.msf	Msf
	20190927_Ribosomes_1.raw	Raw
	20190927_Ribosomes_2.msf	Msf
	20190927_Ribosomes_2.raw	Raw
	20190927_Ribosomes_Full2.pep.xml	Pepxml

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Publications

RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1.

Matsuo Yoshitaka Y Tesina Petr P Nakajima Shizuka S Mizuno Masato M Endo Akinori A Buschauer Robert R Cheng Jingdong J Shounai Okuto O Ikeuchi Ken K Saeki Yasushi Y Becker Thomas T Beckmann Roland R Inada Toshifumi T

Nature structural & molecular biology 20200323 4

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal t ...[more]

PMID: 32203490

Similar Datasets

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.