Figure 1.
(A) Schematic drawing of the fru genomic in Ae. aegypti (not to scale). We renamed the five common Aeafru exons as C1, C2, C3, C4 and C5 and the zinc finger type C encoding exon as zC. Ensembl exon names (Ensembl id: AAEL006301) are shown in parentheses above exons. Translational start (ATG) and stop (TGA, TAA) sites are marked. The exons C1 and C2 encode the BTB domain; the exons C3, C4 and C5 encode the connecting region; the terminal exon zC encodes the type C zinc-finger domain. Exon P1 (named exon S in Demir et al., 2005) is divided in two sub-regions, a male- (P1-m in blue) and a female-specific (P1-f in pink) portion, which are alternatively spliced in a sex-specific mode. This regulation results in different 5′ encoding regions with a male-specific ATG signal in exon P1-m and multiple stop signals in exon P1-f that lead in female to the use of a non-sex-specific ATG signal in exon C1. Exon P2 is present in both sexes and encodes a short non-sex-specific N-terminal box 8 aa long. (B) RT-PCR amplifications of Aeafru sex-specific and common cDNA fragments on sexed adult Ae. aegypti mosquitoes. Primers used in the PCR amplifications are indicated as short red arrows in Fig. 1A.
Figure 2.
Multiples sequence alignment of the FRU isoforms.
Protein sequence alignment of the fru isoforms of D. melanogaster, An. gambiae and Ae. aegypti. The conserved BTB domain and zinc finger domains are boxed in grey. Bold letters indicate amino acid identity among Drosophila, Anopheles and Aedes or between two of them. Intron positions are indicated by solid triangles and position of 3′ alternative splicing site is indicated by AS triangles. Gaps were introduced in the alignments to maximize similarity. The sequences are divided into: (A) a male-specific N-terminal portion encoded by AeaP1 transcripts; (B) an alternative common N-terminal portion encoded by AeaP2 transcripts (the N-terminal extension of the Aedes FRUP2-C isoform is similar to the Drosophila FRU isoforms encoded by transcripts derived from the P3 promoter; in these Dmfru transcripts an ATG signal, located upstream the ATG present in the exon C1, leads to the in frame insertion of a short conserved amino acid box in both species); (C) a portion, common to males and females, including the BTB domain, the connector region and the zinc finger type C domain; (D) putative in silico identified zinc-finger type A and B domains of Ae. aegypti aligned with the homologous domains of D. melanogaster and An. gambiae.
Figure 3.
Comparative scheme of D. melanogaster, Ae. aegypti and An. gambiae fru-P1 genomic structure.
Due to its complex structure, with multiple promoters and 5′ and 3′ alternative splicing, we compare the homologous portion of fru genes, starting with the sex-specific regulated region and ending with the ZnF-C domain encoding exon. fru-P1 common (but encoding the male-specific N-terminus) and female-specific exons are represented as blue boxes and pink boxes, respectively. Green boxes represent the non-sex-specific exons encoding BTB domain and connector region of FRU proteins while terminal grey boxes represent the ZnF-C domain encoding exons. White rectangles represent TRA/TRA-2 binding sites. The Drosophila fru corresponding region spans 98 Kb and is organized in 7 exons and 6 introns, with 6 common exons, preceded by the sex-specific regulated region with a male-specific and a female-specific exons. DmfruMC translation initiates at the ATG within exon P1-m and terminates within the ZnF-C encoding exon C, while in the case of DmfruC translation initiates at the ATG within the BTB encoding exon C1 and terminates within at the same stop signal in exon ZnF-C.
Figure 4.
Phylogenetic and molecular evolution analyses.
(A) NJ and MP consensus trees based on nucleotide alignment of the BTB encoding region of the fru gene of different insect species. (B) Diagram showing the BTB and the connector domain of the fru gene in four mosquito species. The dN, dS and dN/dS (ω) values for each domain are reported below the scheme.
Figure 5.
Developmental expression analysis of the Aeafru gene.
(A) Aeafru gene, transcripts and protein isoforms. Transcripts derived from a putative AeaP1 promoter consist of seven exons with six introns and are alternatively spliced. The transcripts derived from promoter AaeP2 consist of seven exons and six introns and share with AaeP1 ones the common exons C1-C2-C3-C4-C5 and the zinc-finger exon zC but have the upstream exon P2 with and an alternative ATG signal which use led to the translation of AeaFRUP2-C isoform. The translation of all isolated Aeafru-C isoforms terminates in both sexes at the stop codon (TAA) in the zinc-finger exon zC. Primers used in the following amplifications are indicated as short red arrows. (B) Aedes aegypti ribosomal gene rp49 positive control. (C) Aeafru P1 and P2 developmental expression patterns. (D) Aeafru expression pattern on single sexed larval samples. Sexing of samples was performed using Aeadsx primer pair described in [42], which produces a unique amplification signal of 0.5 Kb in the male sample and two amplification signals, of 1.5 and 1.0 Kb, in the female sample. These signals correspond to Aeadsx gene sex-specifically spliced transcripts. E = 0–36 h old embryos; L12 = early larvae; L34 = late larvae; P = pupae; M = adult males; F = adult female. All samples are composed of mixed sexes except for larvae and pupae samples of panel C, which are constitute of single sexed late larvae or pupae.
Figure 6.
Sliding window analysis of MEME identified motifs.
Schematic diagram of the sliding window analysis performed on (A) Aeadsx and (B) Aeafru sex-specifically regulated regions. Both regions are represented in scale and aligned with the corresponding sliding window graph. Each sliding window is 100 bp long and overlaps for 50 bp with the following and preceding sliding windows; each x axis position represent the nucleotide position of the centre of the sequence window. Scores (y axis) are calculated as described in Methods Section and are expressed in log10 of the total sliding window score multiplied for 2. Motif legend is reporter below graphs.
Figure 7.
Phylogenetic and molecular evolution analyses of tra-2 in mosquitoes.
(A) NJ consensus tree based on nucleotide alignment of the RRM+Linker encoding regions of the tra-2 gene of different insect and non insect species. (B) Table with the percentage of identity of the same nucleotide sequences analysed in the NJ tree. (C) WebLogo consensus sequences of TRA/TRA-2 binding sites of the indicated species. Only for mosquito species is it not possible to define a clear consensus sequence.