Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5′ leader regions

Abstract

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome

“…The precise step-by-step mechanism leading to these changes, however, remains to be fully elucidated. DMDAPatA and hippuristanol, which have different mechanisms of eIF4A inhibition compared to rocaglates ( Naineni et al, 2021 ; Shen and Pelletier, 2020 ; Steinberger et al, 2020 ), replicated at least some key reprogramming effects. Hippuristanol inhibits the interaction of both free and eIF4F-bound eIF4A with RNA ( Bordeleau et al, 2006b ), while pateamine is more similar to the rocaglates, increasing the target’s interaction with RNA, making it less available to take part in the complex ( Bordeleau et al, 2006a ).…”

Expression and Functional Roles of Eukaryotic Initiation Factor 4A Family Proteins in Human Cancers

“…Previous studies have shown that the natural marine product, elatol, inhibits eIF4A1, providing a highly promising target for cancer therapy (Peters et al, 2018). Furthermore, hippuristanol, silvestrol, pateamine A, and oxo-aglaiastatin all target eIF4A1 (Itoua Maïga et al, 2019;Naineni et al, 2020;Steinberger et al, 2020). Rocaglates have been shown to possess potent antineoplastic activity both in vivo and in vitro by enhancing mRNA binding to both eIF4A1 and eIF4A2 (Chu et al, 2019).…”

DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy

“…Hippuristanol, as a polyoxygenated steroid, selectively inhibits eIF4A through binding to its C-terminal domain [ 119 ]. Hippuristanol suppresses the RNA-binding activity of eIF4A by locking eIF4A in a closed conformation, but does not prevent its ATP-binding activity ( Table 2 ) [ 72 , 120 ].…”