N6-adenosine (m6A) mRNA methylation is required for Tribolium castaneum development and reproduction

Abstract

Gene expression is regulated at various levels, including post-transcriptional mRNA modifications, where m⁶A methylation is the most common modification of mRNA. The m⁶A methylation regulates multiple stages of mRNA processing, 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 role of m⁶A modification in insect development. RNA interference (RNAi)-mediated knockdown of genes coding for m⁶A writers (m⁶A methyltransferase complex, depositing m⁶A to mRNA) and readers (YTH-domain proteins, recognizing and executing the function of m⁶A) was conducted. Knockdown of most writers during the larval stage caused a failure of ecdysis during eclosion. The loss of m⁶A machinery sterilized both females and males by interfering with the functioning of reproductive systems. Females treated with dsMettl3, the main m⁶A methyltransferase, laid significantly fewer and reduced-size eggs than the control insects. In addition, the embryonic development in eggs laid by dsMettl3 injected females was terminated in the early stages. Knockdown studies also showed that the cytosol m⁶A reader, YTHDF, is likely responsible for executing the function of m⁶A modifications during insect development. These data suggest that m⁶A modifications are critical for T. castaneum development and reproduction.