Abstract
SummaryTranslating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Highlights
The rate of elongation during translation of mRNA by a ribosome is non-uniform
Similar to Slh1 knockout in yeast (Matsuo et al, 2017), knockdown of ASCC3 in cultured mammalian cells allowed increased readthrough of (KAAA)21, a stalling sequence encoded by 21 AAA Lysine codons (Figures 1A and 1B)
Like ZNF598 (Garzia et al, 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al, 2017), ASCC3 is needed to terminally abort translation at a poly(A)-mediated stall similar to other stalls (Hashimoto et al, 2020)
Summary
The rate of elongation during translation of mRNA by a ribosome is non-uniform. Numerous causes of site-specific ribosome slowing have been described. Some slowdowns are beneficial and act to improve dynamic processes, such as co-translational protein folding (Stein et al, 2019), protein targeting to an organelle (Pechmann et al, 2014), and mRNA localization (Yanagitani et al, 2011). Other slowdowns, such as those triggered by a damaged mRNA, are pathological (Doma and Parker, 2006; Joazeiro, 2017). Considerable attention has turned to understanding how cells detect collisions, how detection is converted into multiple downstream responses, and how the collided ribosome complex is resolved
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