Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

Abstract

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs1-4. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5′ UTRs that depend strongly on eIF4A-mediated unwinding5. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA1,2,6. Here, we show that secondary structure in 5′ UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.

Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma

“…These compounds increase the affinity between eIF4A and mRNA and specifically clamp eIF4A onto polypurine sequences in mRNA 5′UTR, thus blocking ribosome subunit scanning and reducing protein expression from transcripts bearing the rocaglamide A–eIF4A target sequence (35). The compound CMLD010509 is a potent and selective translation inhibitor through an eIF4E phosphorylation–independent mechanism (15).…”

Slowed decay of mRNAs enhances platelet specific translation

“…34,36 To comprehensively test how platelet activation affects translation of specific mRNAs, we performed ribosome profiling on platelets activated with thrombin for 60 minutes at 37°C. To estimate global protein synthesis, we quantified the increase in overall cytosolic ribosome footprints after normalization to footprints derived from mitochondrial ribosomes, 64,65 which are entirely distinct from the cytosolic translational machinery and are continuously synthesized in platelets. 66 These analyses show there is a global increase in ribosome occupancy on cytoplasmic mRNAs, consistent with increased translational output (Figure 1E-F; gray vs blue transcripts).…”

Targeting RNA helicases in cancer: The translation trap

“…Translation initiation could be rescued with recombinant eIF4A after Hippuristanol treatment, showing that Hippuristanol specifically mediates it effect through eIF4A inhibition[19]. mRNAs with a long or complex 5′UTR are most affected by Hippurastinol treatment[78]. …”

“…indicated that in contrast to Hippurastinol, a complex 5′UTR was only a minor determinant of Rocaglamide A (RocA) efficacy. Instead they found RocA to clamp eIF4A on mRNAs that have short polypurine sequences in their 5′UTR, hereby putting up a roadblock for ribosomal scanning[78] (Figure 4). Unfortunately, RocA was not compared directly to Silvestrol in this study, and it remains to be determined whether the specificity for polypurine mRNA sequences is a feature of all rocaglates.…”

“…Pateamine A (PatA) stimulates RNA binding activity of free eIF4A (eIF4A F )[79], most likely hereby sequestering it and perturbing the interactions with other translation initiation factors[81]. Of the family of Rocaglates, Rocaglamide A (RocA) clamps eIF4A on mRNAs that have short polypurine sequences in their 5′UTR, hereby putting up a roadblock for ribosomal scanning[78]. Silvestrol, one of the most studied rocaglates is thought to sequester eIF4A from the eIF4F complex by its increased RNA affinity[23, 83], whether it also has a polypurine specificity remains to be determined.…”