A gene is transcribed into mRNA in

A recent study has discovered that the common
mRNA modification, m6A, is required for alternative splicing of Drosophila
Sex lethal, the gene responsible for sex determination.

 

The effects of RNA
modifications on gene expression have been less well understood compared to the
effects of DNA and chromatin modifications. However, it has been discovered
that N6-methyladenosine (m6A) is the most prevalent
modification in mRNA (4). Suggestions that m6A regulates alternative
splicing in pre-mRNA introns led to Haussman et al investigating whether m6A
is required for alternative splicing of the Drosophila sex determination factor,
Sex lethal (Sxl). The Sxl gene is transcribed into mRNA in males and females,
however alternative splicing results in a functional protein only being
produced in the female. This is because the female Sxl prevents inclusion of
the male-specific alternative exon containing a stop codon, which would
otherwise prevent Sxl protein synthesis (5).

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The authors began by
producing a null allele of Drosophila Ime4, a nuclear protein of the m6A
methylosome. This Ime4null allele produces viable and fertile, yet
flightless flies. The RNA-binding protein Sxl positively auto-regulates
expression of itself and its target transformer (tra) through alternative
splicing, leading to female differentiation. Sxl also suppresses translation of
male-specific lethal 2 (msl-2), to prevent dosage compensation in females (3).
Consequently, the authors demonstrated that removal of the maternal m6A,
together with crossing Ime4null females with Sxl null males, results
in non-viable females due to inadequate suppression of msl-2 expression. This
results in upregulation of transcription on the X chromosome because of reduced
Sxl levels, proving that m6A functions in Sxl alternative splicing.

 

To further understand
Sxl alternative splicing defects in Ime4null females, Haussman and
colleagues examined splice junction reads from RNA-sequencing. They discovered
a significant increase in the presence of the male-specific Sxl exon in Ime4null
females due to a reduction of female splicing, further supporting the role of m6A
in Sxl alternative splicing. The authors next used an in vitro m6A
methylation assay to map m6A sites in the intron of Sxl, identifying
m6A in the proximity of Sxl-binding sites. Single amino acid
substitutions of the methylosome components female-lethal (2)d (fl(2)d) and
virilizer (vir), impeded Sxl recruitment, leading to defective Sxl
auto-regulation and inclusion of the male-specific exon. This further
demonstrated the role of the m6A methylosome in Sxl alternative
splicing.

 

m6A is
decoded by YTH domain proteins, including YT521-B and CG6422, and consequently
Haussman and colleagues tested to see whether these proteins decode m6A
in Sxl mRNA. They discovered that nuclear YT521-B can switch Sxl alternative
splicing to the female mode and in addition, YT521-B demonstrated increased
binding to m6A-containing RNA. The authors next examined the
Drosophila strain YT521-BMI02006, containing a transposon in the
first intron disrupting YT521-B. Consistent with the Ime4null flies,
this YT521-B allele also produced viable yet flightless flies, with the Sxl
splicing defect, suggesting YT521-B is the main mediator of m6A-depedendent
processes. Consequently, Haussman and colleagues demonstrated that YT521-B is
the main nuclear protein for assisting Sxl in preventing the inclusion of the
male-specific exon by binding to nearby intronic m6A sites, as
illustrated in Figure 1.

 

Figure
1 – YT521-B assists m6A in
regulating alternative splicing to produce the female splicing pattern. m6A
and YT521-B skip the male-specific exon containing a stop codon, allowing
production of a transcript encoding the Sxl protein.

 

This study by Haussman
and co-workers therefore demonstrates a function for m6A, the ‘fifth
nucleotide’ in regulating alternative splicing to determine the sex of
Drosophila fruit flies. It particularly highlights the importance of m6A
and its reader YT521-B in supporting this gene regulatory switch, which has been
confirmed by further studies (2). However, it remains unclear how m6A
and YT521-B interact with other components of the Sxl splicing machinery. One
suggestion is that YT521-B may interact with specific co-factors to help
mediate different regulatory outcomes of sex differentiation (2). Nonetheless,
this study demonstrates that m6A modification is an ancient
mechanism, with the m6A methylosome playing a key role in human
dosage compensation. Finally, this study demonstrated that a lack of Sxl in
stem cells may result in the development of ovarian cancer. Therefore, this
relationship between the m6A methylosome and regulation of gene
transcription may with future research provide links to human diseases such as
cancer.