N6-Adenosine (m6A) mRNA methylation is required for Tribolium castaneum ecdysis, oogenesis and embryogenesis

Gene expression is regulated at various levels including posttranscriptional mRNA modification, where m6A methylation is the most prevalent modification on mRNA from yeast to mammals. The m6A methylation has been found to regulate most stages of mRNA processes including splicing, export, decay, and translation. How m6A modification is involved in insect development is not well known. We used the red flour beetle, Tribolium castaneum as a model insect to identify the function of m6A modification in insect development. Loss-of-function study was achieved by knocking down m6A pathway players including m6A writers (m6A methyltransferase complex, depositing m6A to mRNA) and readers (YTH-domain proteins, recognizing and executing the function of m6A). By injecting dsRNAs in the newly molted last instar larvae, it was revealed that the knockdown of most writers caused the failure of ecdysis during eclosion. Knocking down one writer RBM15 caused 100% lethality in larval stage, however, it is mediated in a m6A -independent manner by regulating DNA replication. We further injected dsRNAs to the newly molted adults to investigate the function of m6A on insect reproduction. Mating experiments showed that loss of m6A machinery sterilized females but not males. Females treated with dsMettl3, the main m6A methyltransferase, laid significantly less number and reduced size of eggs when compared with the control. Embryo staining using DAPI revealed that the development of eggs laid by female injected with dsMettl3 terminated at early stage of embryogenesis. The cytosol m6A reader, YTHDF, is likely responsible for executing the function of m6A modification in insect development. These data suggest that m6A modification is essential for the T. castaneum embryogenesis, eclosion, and female fertility.