Dataset Information

Znf598-mediated Rps10/eS10 Ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

ABSTRACT: The ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ubiquitination of the ribosomal proteins plays a pivotal role in translational control and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To study ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. Combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis, we analyzed ribosome ubiquitination during zebrafish development. Our data showed that ubiquitination of ribosomal proteins dynamically changed as development proceeded. We further revealed that Znf598, an E3 ubiquitin ligase that triggers RQC, contributed to the ribosome ubiquitination during zebrafish development. LC-MS/MS analysis and immunoblotting analysis identified lysines 139 of ribosomal protein Rps10/eS10 as pivotal ubiquitination sites on the ribosome during development. Finally, we demonstrated that an Rps10 K139/140R mutation reduced overall ribosome ubiquitination pattern. Collectively, these results reveal dynamics and complexity of ribosome ubiquitination in zebrafish development.

ORGANISM(S): Danio Rerio

SUBMITTER: Yuichiro Mishima

PROVIDER: PXD039560 | JPOST Repository | Wed Oct 11 00:00:00 BST 2023

REPOSITORIES: jPOST

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	210309_KI_Zebra_Mishima-lab_158_0h_rep1.raw	Raw
	210309_KI_Zebra_Mishima-lab_158_0h_rep2.raw	Raw
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Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development.

Ugajin Nozomi N Imami Koshi K Takada Hiraku H Ishihama Yasushi Y Chiba Shinobu S Mishima Yuichiro Y

RNA (New York, N.Y.) 20230926 12

The ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ri ...[more]

PMID: 37751929

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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.