A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells

Abstract

AbstractMono(ADP-ribosyl)ation (MARylation), a post-translational modification (PTM) of proteins, is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and an integral component of the ribosome, is MARylated by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. We mapped and confirmed the sites of MARylation, which occur on three acidic residues within blades 4 and 5 of β-propeller domain of RACK1, a chaperone that shuttles and anchors proteins where needed. Site-specific MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 to stress granules with key components, such as G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1 to dissociate the stress granules and return RACK1 and the 40S ribosomal subunit to the cytoplasm, allowing for a restoration of translation. Collectively, our results highlight the discovery of a PARP14/TARG1-regulated RACK1 MARylation cycle that controls stress granule assembly and disassembly in ovarian cancer cells.