Dm Ime4 depletion affects permeability barrier and Chic function in Drosophila spermatogenesis

Abstract

Adenosine methylation of messenger RNA at the N6 position (m6A) is a non-editing modification that can affect several aspects of mRNA metabolism. Dm Ime4, also known as METTL3, MTA, and MTA-70 in other organisms, is the catalytic subunit of the methyltransferase complex that adds this modification. Dm ime4 is evolutionarily conserved and essential for development in metazoans and plants. Because of its pleiotropic effects, it has been difficult to establish the main reason why embryonic arrest occurs in plants, mice, and zebrafish. Using a strategy that depletes Dm Ime4 specifically in the somatic cyst cells of Drosophila testes without affecting essential functions in development, our lab has found that Dm Ime4 may potentially regulate splicing of profilin (chic) mRNA, the message for an essential and evolutionarily conserved protein mainly known for its function in actin polymerization. One of the lesser known roles for Chic is its requirement for establishment and maintenance of the somatic cyst-cell permeability barrier in Drosophila spermatogenesis. Chic and Dm Ime4 colocalize and are abundant in somatic cyst cells throughout spermatogenesis. Upon selective depletion of Dm Ime4, we observe significant reduction of Chic protein levels and malfunction of the permeability barrier. We have found that chic mRNA contains intronic Dm Ime4 binding sites that can form the hairpin structures required for recognition by the methyltransferase complex. Our data show that the reduced levels of Chic protein observed in Dm ime4 somatic cyst-cell knockdowns could be the result of aberrant splicing of its mRNA. In turn, low levels of Chic are known to affect the function of the somatic permeability barrier, leading to germline death and the reduced fertility observed in Dm ime4 knockdown males. We propose that Dm Ime4 may regulate chic in other developmental contexts and in other organisms, including mice and humans. Chic is an essential protein that is evolutionarily conserved, and establishment and maintenance of cell barriers and domains are important strategies used in metazoan development. Taken together, our findings define a framework to investigate specific functions of Dm Ime4 and its homologs in multicellular organisms by bypassing its pleiotropic requirement in early developmental stages.