m6A modification determines female-specific alternative splicing in Drosophila.
N6-methyladenosine (m6A) is a common modification in mRNA which affects alternative splicing of the Sex-lethal (Sxl). Haussmann et al. demonstrate the requirement of m6A and YT521-B for the expression of the alternative splicing of Sxl specific to females and how this contributes to the suppression of dosage compensation1.
Sex determination in Drosophila is based on the ratio of sex chromosomes to autosomes. If the ratio equals 1, i.e. 2X chromosomes are present, the fly is female via the activation of Sxl gene. Whereas, if the ratio is 0.5, i.e. a single X chromosome is present, the fly is male and Sxl gene is not activated. Therefore, Sxl is a significant factor in determining the sex of Drosophila. The Sxl protein binds to the pre-mRNA of transformer and affects its splicing2. Overall, leading to female-specific physical characteristics and prevention of dosage compensation1. The modification, m6A is caused by the methylosome which is a protein complex that methylates adenosines within the mRNA. The methylosome in flies contains METTL14, Ime4 (Inducer of meiosis 4), KAR4 (Karyogamy protein 4), Female-lethal (2)d (Fl(2)d) and Virilizer (Vir)3.The biological effects of this alteration are dependent on protein readers such as YT521-B; which binds to m6A and mediate its effects on alternative splicing4.
The findings from Haussmann et al. demonstrates that the m6A modification is necessary for the determination of sex in Drosophila and the suppression of dosage compensation in female flies1. The results of a m6A-antibody enrichment show that m6A is easily identified in poly(A) mRNA but is absent in flies which lack Ime4. Furthermore, Ime4-null flies are fertile and viable, but flightless and have a greater chance of being male. This is illustrated by the 60% decrease in the number of females present compared to males1. This can be explained by the requirement of Ime4 as a component of the methylosome that causes m6A modification within mRNA; which is essential for the alternative splicing of Sxl that is specific to female flies. This is important as Sxl both determines the physical characteristics of the female fly and decreases the expression of male-specific lethal-2 which causes dosage compensation in males. Maternal m6A factors are required to completely supress dosage compensation and removal of these can reduce female viability by 13%.
Ime4-null mutants illustrate there are no substantial differences in global alternative splicing compared to wild-type flies. However, there were decreases in alternative splicing events in the alternative first exon and mutually exclusive exon1. 33% of the alternatively spliced genes were found within nervous tissue which contains a greater concentration of Ime4.This suggests that m6A impacts neuronal function and behaviour of flies5. Additionally, Ime4 may influence gene expression as it was found to be located at transcription sites. Some of these affected genes resulted in a decrease in the expression of proteins involved in oxidative phosphorylation and affected overall metabolism1.
YT521-B is a nuclear protein with YTH domain that decodes m6A sites in Sxl mRNA in male S2 cells.YT521-B causes the alternative splicing of Sxl to become female-specific. The YTH domain of YT521-B showed increased binding with RNA that contained m6A within an in vitro experiment. Furthermore, YT521-B is also found at the transcription sites in vivo. m6A and YT521-B work in conjunction to prevent the addition of the male exon during alternative splicing of Sxl. Thus, contributing to female-specific alternative splicing; which leads to female physical characteristics and suppression of dosage compensation. The absence of YT521-B has a similar effect to Ime4-null mutants, in which viable but flightless flies are produced1. This suggests that YT521-B is an essential factor that is required to allow effects of m6A marks in close vicinity binding site of Sxl to be observed.
m6A alters alternative splicing which influences gene expression and sex determination in Drosophila. mRNA modification is highly significant and needs to be further studied to provide a comprehensive understanding of the processes. This includes determining the significance of each component on the function of methylosome and how different protein readers interact with m6A; for example, the different specificities of certain readers for m6A and how they may compete to influence the effects observed.