N6-Adenosine (m6A) mRNA methylation is required for Tribolium castaneum ecdysis and female reproductio

Gene expression is regulated at various levels, including post-transcriptional mRNA modifications, where m6A methylation is the most prevalent modification of mRNA from yeast to mammals. The m6A methylation was found to regulate every stage of mRNA life, including splicing, export, decay and translation. The role of m6A mRNA modifications in insect development is not well studied. The red flour beetle, Tribolium castaneum, due to its sensitivity to RNAi, is a promising model to study insect biology. To study the function of m6A , the orthologs of writers (m6A methyltransferase complex, depositing m6A to mRNA) and readers (YTH-domain proteins, recognizing m6A and executing function) were identified by searching the genome of T. castaneum. Knockdown of most writers (Mettl3, WTAP, Flacc, Virilizer) by injecting dsRNA into the last instar larvae caused a block in pupal ecdysis. To study the effect of m6A on reproduction, dsRNAs targeting these genes were injected into newly emerged adults. Mating experiments revealed that loss of m6A machinery sterilized females but not males. Knockdown of Mettl3 (a core enzyme of m6A writer complex) in females reduced the number of eggs laid, and most of the eggs laid failed to hatch. The oocytes in females treated with dsMettl3 were smaller compared to those in controls treated with dsmalE. The staining of embryos using DAPI revealed that the development was blocked at the early embryonic stage. These data suggest that m6A methylation is essential to T. castaneum ecdysis and female reproduction.