Limits...
Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti.

Salvemini M, Mauro U, Lombardo F, Milano A, Zazzaro V, Arcà B, Polito LC, Saccone G - BMC Evol. Biol. (2011)

Bottom Line: The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation.Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner.Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Genetics and Molecular Biology, University of Naples Federico II, Italy.

ABSTRACT

Background: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.

Results: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.

Conclusions: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

Show MeSH

Related in: MedlinePlus

Putative cis-acting elements of the Aeadsx gene. The distribution of the putative cis-acting elements in the sex-specifically spliced region of Aeadsx is reported. Black lowercase letters indicate intronic regions. Dark purple letters indicate the female-specific 5a and 5b exons. Translational stop codons are reported in red uppercase letters. The putative polyadenylation signal of exon 5a is underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045327&req=5

Figure 6: Putative cis-acting elements of the Aeadsx gene. The distribution of the putative cis-acting elements in the sex-specifically spliced region of Aeadsx is reported. Black lowercase letters indicate intronic regions. Dark purple letters indicate the female-specific 5a and 5b exons. Translational stop codons are reported in red uppercase letters. The putative polyadenylation signal of exon 5a is underlined.

Mentions: We then used two other primer pairs to extend the RT-PCR expression analysis. The dsx3/dsx4 pair led to amplification of the expected female-specific 0.4-kb long cDNA product in adult females (see Figure 5B.2). This cDNA product was amplified at larval and pupal stages, confirming the previous data that showed the presence of exon 5a during this developmental period. However, the 0.4-kb cDNA fragment was also amplified at embryonic and 1st/2nd instar larval stages, indicating that exon 5a is present in mRNAs earlier than previously shown (see Figure 5B.2 and 5B.5). A possible explanation of this apparently contrasting data is that there is a third alternative mRNA in which exon 5a is present but exon 5b and exon 6 are not. This mRNA could be generated by alternative polyadenylation at the 3' UTR of exon 5a. A northern blot analysis of total RNA from sexed adult mosquitoes showed the presence of a third female-specific Aeadsx transcript, offering support to this explanation (Figure 5C). The two mRNAs including exon 5a are different in the 3' UTR but potentially encode the same DSXF1 protein. Furthermore, a putative polyadenylation signal (AATAGA) was identified in silico 333 bp downstream of the stop codon present in exon 5a (Figure 6). Interestingly, the Apis mellifera dsx homologue produces two female-specific transcripts using two alternative polyadenylation signals, and possibly as in Aedes, they both encode the same DSXF protein [21].


Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti.

Salvemini M, Mauro U, Lombardo F, Milano A, Zazzaro V, Arcà B, Polito LC, Saccone G - BMC Evol. Biol. (2011)

Putative cis-acting elements of the Aeadsx gene. The distribution of the putative cis-acting elements in the sex-specifically spliced region of Aeadsx is reported. Black lowercase letters indicate intronic regions. Dark purple letters indicate the female-specific 5a and 5b exons. Translational stop codons are reported in red uppercase letters. The putative polyadenylation signal of exon 5a is underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045327&req=5

Figure 6: Putative cis-acting elements of the Aeadsx gene. The distribution of the putative cis-acting elements in the sex-specifically spliced region of Aeadsx is reported. Black lowercase letters indicate intronic regions. Dark purple letters indicate the female-specific 5a and 5b exons. Translational stop codons are reported in red uppercase letters. The putative polyadenylation signal of exon 5a is underlined.
Mentions: We then used two other primer pairs to extend the RT-PCR expression analysis. The dsx3/dsx4 pair led to amplification of the expected female-specific 0.4-kb long cDNA product in adult females (see Figure 5B.2). This cDNA product was amplified at larval and pupal stages, confirming the previous data that showed the presence of exon 5a during this developmental period. However, the 0.4-kb cDNA fragment was also amplified at embryonic and 1st/2nd instar larval stages, indicating that exon 5a is present in mRNAs earlier than previously shown (see Figure 5B.2 and 5B.5). A possible explanation of this apparently contrasting data is that there is a third alternative mRNA in which exon 5a is present but exon 5b and exon 6 are not. This mRNA could be generated by alternative polyadenylation at the 3' UTR of exon 5a. A northern blot analysis of total RNA from sexed adult mosquitoes showed the presence of a third female-specific Aeadsx transcript, offering support to this explanation (Figure 5C). The two mRNAs including exon 5a are different in the 3' UTR but potentially encode the same DSXF1 protein. Furthermore, a putative polyadenylation signal (AATAGA) was identified in silico 333 bp downstream of the stop codon present in exon 5a (Figure 6). Interestingly, the Apis mellifera dsx homologue produces two female-specific transcripts using two alternative polyadenylation signals, and possibly as in Aedes, they both encode the same DSXF protein [21].

Bottom Line: The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation.Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner.Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Genetics and Molecular Biology, University of Naples Federico II, Italy.

ABSTRACT

Background: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.

Results: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.

Conclusions: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

Show MeSH
Related in: MedlinePlus