Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5'...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Hippuristanol (Hipp) is a natural product that inhibits protein synthesis by targeting eukaryotic initiation factor 4A (eIF4A).
• eIF4A is a DEAD-box RNA helicase crucial for ribosome recruitment to mRNA, and its inhibition is a potential therapeutic strategy for cancer and viral infections.
• mRNA dependencies on eIF4A are influenced by the secondary structure of their 5' leader regions.

Purpose of the Study:

• To identify functional EIF4A1 alleles resistant to Hippuristanol using a CRISPR/Cas9-based variomics screen.
• To link the translation-inhibitory and cytotoxic effects of Hippuristanol to eIF4A1 target engagement.
• To elucidate the structure-activity relationships of eIF4A-dependent mRNAs.

Main Methods:

• CRISPR/Cas9-based variomics screening to identify Hippuristanol-resistant EIF4A1 alleles.
• Genome-wide translational profiling in the presence and absence of Hippuristanol.
• Validation studies to analyze structure-activity relationships of eIF4A-dependent mRNAs.

Main Results:

• Identification of functional EIF4A1 alleles conferring resistance to Hippuristanol.
• Confirmation of Hippuristanol's translation-inhibitory and cytotoxic effects being linked to eIF4A1 target engagement.
• Determination that mRNA 5' leader length, secondary structure, and cytosine content define Hippuristanol-dependent mRNAs.

Conclusions:

• Hippuristanol's mechanism of action involves binding to eIF4A, locking it in a closed conformation, and inhibiting RNA binding.
• The study provides insights into the structural features of mRNAs that dictate their dependency on eIF4A for translation.
• Findings support the therapeutic potential of targeting eIF4A, with Hippuristanol as a valuable chemical probe.