The deubiquitylase USP9X controls ribosomal stalling.

Anne Clancy,Stephanos Ioannidis,Simon Davis,Sunkyu Kim,David Komander,Axel Behrens,Benedikt M Kessler,Crystal Mckinnon,Michael J Clague,Hannah Elcocks,Steven Mischke,Claire Heride,Marie Katz,Weiping Wang,Neil P Jones,Justin A Caravella,Katherine J Kayser-Bricker,Tim Hammonds,Bruce Follows,Andreas Kallinos,Victoria Smith,Sylvie Urbé,Adán Pinto-Fernández,Shawn P Fessler ,Christopher J Dinsmore ,Jonathan C O’connell

doi:10.1083/jcb.202004211

Abstract

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

Highlights

Prompt sensing and resolution of aberrant protein translation is essential for the maintenance of protein homeostasis
Two lines of argument suggest that this is not an effect on transcription: (1) endogenous ZNF598 mRNA levels are similar between the two cell lines (Fig. 2 B), and (2) levels of exogenous HA-ZNF598 expression that is driven by a non-native promoter are diminished in transfected cells (Fig. 2 C)
USP9X is for the most part a nonessential DUB family member that is expressed at relatively high levels (Behan et al, 2019; Clague et al, 2015)

Summary

Introduction

Prompt sensing and resolution of aberrant protein translation is essential for the maintenance of protein homeostasis. If a ribosome stalls during translation, it risks being rear-ended by a trailing ribosome This collision generates a stable di-ribosome complex with a defined structure, which is resolved by the engagement of a dedicated machinery (Juszkiewicz et al, 2018; Collart and Weiss, 2020). In such cases, the E3-ligase ZNF598 ubiquitylates 40S complexes at specific sites on eS10 and uS10 subunits at the di-ribosome interface (Garzia et al, 2017; Juszkiewicz et al, 2018; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al, 2017). A recent report has provided evidence that the E3-ligases Makorin 1 (MKRN1) and Makorin 2 (MKRN2) may complement the activity of ZNF598 in the ribosomal quality control pathway by promoting the initial stalling of the leading ribosome as it encounters a polyA tract (Hildebrandt et al, 2019)

Methods

Results

Conclusion