Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized

Abstract

Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role and fate of ubiquitylated proteins remain unclear. We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation, respectively, suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes. We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation. Critically, cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway.

EDF1 coordinates cellular responses to ribosome collisions

“…We performed a similar experiment in ΔEDF1 cell lines ( Figure 2—figure supplement 2C–2D for characterization of knockout lines) under physiological conditions that induce ribosomal collisions ( Garshott et al, 2020 ; Wu et al, 2020 ). Parental and three different clonal populations of ΔEDF1 cells were left untreated or subjected to UV irradiation.…”

The ASC-1 Complex Disassembles Collided Ribosomes

“…The steps preceding ASCC are potentially reversible: collisions can be reversed if translation resumes and ubiquitinated 40S can be deubiquitinated ( Garshott et al., 2020 , Meyer et al., 2020 ). This means that translation is abandoned only when a collision is sufficiently long-lived to recruit ZNF598, and the 40S-ubiquitin mark lingers long enough to allow ASCC function.…”

Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

“…A time course of ZNF598 ubiquitination of ribosomes in vitro at close to physiologic concentrations indicate that it takes at least ~2–5 min to accumulate appreciable 40S ubiquitination ( Juszkiewicz and Hegde, 2017 ). Because there are likely to be deubiquitinase(s) that can act on the ribosome ( Garshott et al, 2020 ), 40S ubiquitination is not the commitment step to trigger RQC. Instead, ubiquitinated eS10 is needed for the ASC-1 complex (ASCC) to act ( Juszkiewicz et al, 2020 ; Matsuo et al, 2020 ).…”