OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation

Abstract

Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression.

OLA1, a Translational Regulator of p21, Maintains Optimal Cell Proliferation Necessary for Developmental Progression

“…Whereas most null mutations of the YchF homologue in yeasts are nonlethal (7), inactivation of TcYchF by RNA interference (RNAi) in trypanosomes (Trypanosoma cruzi) inhibited the protozoan's growth (4). We (6) and others (8) reported that OLA1 knockdown (KD) in human cells under normal culture conditions had, depending on the origin of the cell lines used, either a negative effect or no effect on cell proliferation. However, under multiple cellular stresses, including oxidative stress, OLA1 KD promoted cell survival (9) by "gating" ISR and the expression of downstream proapoptotic factors, such as CHOP (6).…”

“…We (6) and others (8) reported that OLA1 knockdown (KD) in human cells under normal culture conditions had, depending on the origin of the cell lines used, either a negative effect or no effect on cell proliferation. However, under multiple cellular stresses, including oxidative stress, OLA1 KD promoted cell survival (9) by "gating" ISR and the expression of downstream proapoptotic factors, such as CHOP (6). These paradoxical findings suggest that OLA1 plays multiple roles in the regulation of cell proliferation and cell survival.…”

“…The TRAFAC GTPases include not only translation factors and ribosome-associated proteins but also proteins that are involved in signal transduction, intracellular transport, and stress responses (1,2). The YchF/OLA1 proteins are highly conserved from bacteria to humans, and unlike other Obg family members, they possess both GTPase and ATPase activities (4)(5)(6). We recently revealed that human OLA1 is a eukaryotic initiation factor 2 (eIF2)-regulatory protein that suppresses protein synthesis and enhances the integrated stress response (ISR) by binding eIF2 and interfering with the formation of the eIF2-GTP-Met-tRNAi Met (ternary) complex (6).…”

“…Immunoblotting analyses were performed as previously described (6). Briefly, equal amounts of protein were loaded in a 4 to 20% gradient polyacrylamide gel (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane.…”

“…A solution of 100 g/ml of cycloheximide was added to the cells 10 min before cell lysis. Cell lysates were analyzed using polysome profiling as previously described (6). Total RNA was precipitated from the fractions in ethanol and purified using an RNA minikit (Qiagen).…”