Dynamic eIF3-S6 Phase Separation Switch Instructed by m⁶A Modification Drives the Molting of Locusts

Molting is critical for the development and growth of insects. A central juncture in molting is the periodic and coordinated regulation of molting-responsive mRNAs, which are essential for degrading the old cuticle and forming a new one. Knowledge is lacking of how molting-responsive molecules function cooperatively and timely. Here, multi-stage m⁶A modification levels, m⁶A reader condensation, and transcriptome analyses are developed to track the dynamic functional coupling for molting signals. The results showed that the molting-responsive mRNAs are downregulated with the decrease of m⁶A modification levels from pre-molt to the molt stage. The eIF3-S6 is further identified, as an m⁶A reader, can directly bind to EcR A³³⁵ and Cht10 A⁷⁵⁸⁹. The low mRNA m⁶A modification in the molt stage facilitates eIF3-S6 to undergo liquid-liquid phase separation (LLPS) by binding m⁶A-containing EcR/Cht10 into condensed droplets for stabilizing the mRNAs. Disruption of m⁶A modification or breaking phase separation can cause a disorder of the new/old cuticle synthesis/degradation in molting. The disorder results in abnormal and unsuccessful molting, ultimately leading to the death of nymphs. Overall, the data mechanistically highlight an example of how two central machineries, m⁶A modification and LLPS, can accurately and functionally cooperate to optimize molting-responsive gene expression for insect molting.