Monitoring RNA restructuring in a human cell-free extract reveals eIF4A-dependent and eIF4A-independent unwinding activity.

The canonical DEAD-box helicase, eIF4A, unwinds 5 UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to precisely monitor helicase activity in the 5 UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5 UTR-dependent duplex unwinding in the absence or presence of Hippuristanol, a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that roughly 50% of the duplex unwinding activity in the cell-free extract can be attributed to an eIF4A-dependent mechanism, while the remaining 50% of duplex unwinding activity is attributed to an eIF4A-independent mechanism. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.