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Male-specific phosphorylated SR proteins in adult flies of the Mediterranean fruitfly Ceratitis capitata.

Saccone G, Louis C, Zhang H, Petrella V, Di Natale M, Perri M, Salvemini M - BMC Genet. (2014)

Bottom Line: In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx).To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly).We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.

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ABSTRACT
Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.

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SDS-PAGE electropherograms (A-B-C) and immunoblots (D-E-F-G-H) produced with protein extracts from adult males and females of Drosophila, M. domestica and C. capitata. 1) total lysate of males; 2) total lysate of females; 3) 90% ammonium sulfate precipitation supernatant of males; 4) 90% ammonium sulfate precipitation supernatant of females; 5) Mg++ supernatant of males; 6) Mg++ supernatant of females; 7) Mg++ pellets of males; 8) Mg++ pellets of females; P-) Mg++ pellets of C. capitata males without phosphatase treatment; P+) Mg++ pellets of C. capitata males with phosphatase treatment. After transfer to nitrocellulose, blots were incubated with either mAb104 or no primary antibody (as a control; data not shown).
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Figure 1: SDS-PAGE electropherograms (A-B-C) and immunoblots (D-E-F-G-H) produced with protein extracts from adult males and females of Drosophila, M. domestica and C. capitata. 1) total lysate of males; 2) total lysate of females; 3) 90% ammonium sulfate precipitation supernatant of males; 4) 90% ammonium sulfate precipitation supernatant of females; 5) Mg++ supernatant of males; 6) Mg++ supernatant of females; 7) Mg++ pellets of males; 8) Mg++ pellets of females; P-) Mg++ pellets of C. capitata males without phosphatase treatment; P+) Mg++ pellets of C. capitata males with phosphatase treatment. After transfer to nitrocellulose, blots were incubated with either mAb104 or no primary antibody (as a control; data not shown).

Mentions: To search for sex-specific differences in the phosphorylation of major SR splicing factors in D. melanogaster, C. capitata and M. domestica, proteins were extracted from male and female adult tissues. We enriched for SR proteins following a two-step protocol as described in [41]. In Figure 1 whole staining of SR protein enriched extracts as well as immunoblots with the mAb104 antibody are depicted. In all samples we recovered a pattern of enriched SR proteins similar in both males and females (Figure 1A, B and C). Following quantitation of the supernatant proteins and of the soluble proteins after magnesium precipitation from the last purification step, SDS-PAGE separations were performed in parallel duplicates, with one stained with Coomassie Blue (to control for equal loading) and a second one blotted onto a filter. The transfer of proteins extracted from each sex was controlled by reversible Ponceau staining, which is a validated method to assess both equal gel loading and protein transfer [42,43] (Additional file 1 - Figure S1). No sex-specific differences were evident. The filters were used for immunostaing and, on the contrary, showed some sex-specific differences. In Drosophila adult flies, the mAb104 antibody detects 6-8 polypeptides of molecular weights similar to those previously observed in Drosophila Kc cell line [37]. However, the relative amounts appear different in males and females (odd numbers: males; even numbers: females). For instance, the 95 kDa signal is significantly stronger in males, while the 75 kDa signal is more prominent in females (Figure 1D, lanes 7-8). In M. domestica four major phophorylated SR proteins were detected with comparable levels in both sexes (Figure 1E, Figure lanes 7-8). In two immunoblots with samples isolated from two different biological replicates of adult C. capitata flies, we observed that phosphorylated polypeptides were highly enriched only in males (Figure 1, lanes 7 of blots in F and G). Two of the six major SR antigens detected (~75 kDa and ~30 kDa) can also be seen at much lower levels in the female samples in one of the 2 blots (Figure 1, blot in F, lanes 8). As only 0.04% of cell proteins are SR proteins [39] and the mAb104 can detect them only by Mg++ enrichment, we propose that the male-specific C. capitata SR antigens correspond most likely to phosphorylated SR proteins expressed in most of the fly tissues rather then small tissues (such as for example the male germ line).


Male-specific phosphorylated SR proteins in adult flies of the Mediterranean fruitfly Ceratitis capitata.

Saccone G, Louis C, Zhang H, Petrella V, Di Natale M, Perri M, Salvemini M - BMC Genet. (2014)

SDS-PAGE electropherograms (A-B-C) and immunoblots (D-E-F-G-H) produced with protein extracts from adult males and females of Drosophila, M. domestica and C. capitata. 1) total lysate of males; 2) total lysate of females; 3) 90% ammonium sulfate precipitation supernatant of males; 4) 90% ammonium sulfate precipitation supernatant of females; 5) Mg++ supernatant of males; 6) Mg++ supernatant of females; 7) Mg++ pellets of males; 8) Mg++ pellets of females; P-) Mg++ pellets of C. capitata males without phosphatase treatment; P+) Mg++ pellets of C. capitata males with phosphatase treatment. After transfer to nitrocellulose, blots were incubated with either mAb104 or no primary antibody (as a control; data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255826&req=5

Figure 1: SDS-PAGE electropherograms (A-B-C) and immunoblots (D-E-F-G-H) produced with protein extracts from adult males and females of Drosophila, M. domestica and C. capitata. 1) total lysate of males; 2) total lysate of females; 3) 90% ammonium sulfate precipitation supernatant of males; 4) 90% ammonium sulfate precipitation supernatant of females; 5) Mg++ supernatant of males; 6) Mg++ supernatant of females; 7) Mg++ pellets of males; 8) Mg++ pellets of females; P-) Mg++ pellets of C. capitata males without phosphatase treatment; P+) Mg++ pellets of C. capitata males with phosphatase treatment. After transfer to nitrocellulose, blots were incubated with either mAb104 or no primary antibody (as a control; data not shown).
Mentions: To search for sex-specific differences in the phosphorylation of major SR splicing factors in D. melanogaster, C. capitata and M. domestica, proteins were extracted from male and female adult tissues. We enriched for SR proteins following a two-step protocol as described in [41]. In Figure 1 whole staining of SR protein enriched extracts as well as immunoblots with the mAb104 antibody are depicted. In all samples we recovered a pattern of enriched SR proteins similar in both males and females (Figure 1A, B and C). Following quantitation of the supernatant proteins and of the soluble proteins after magnesium precipitation from the last purification step, SDS-PAGE separations were performed in parallel duplicates, with one stained with Coomassie Blue (to control for equal loading) and a second one blotted onto a filter. The transfer of proteins extracted from each sex was controlled by reversible Ponceau staining, which is a validated method to assess both equal gel loading and protein transfer [42,43] (Additional file 1 - Figure S1). No sex-specific differences were evident. The filters were used for immunostaing and, on the contrary, showed some sex-specific differences. In Drosophila adult flies, the mAb104 antibody detects 6-8 polypeptides of molecular weights similar to those previously observed in Drosophila Kc cell line [37]. However, the relative amounts appear different in males and females (odd numbers: males; even numbers: females). For instance, the 95 kDa signal is significantly stronger in males, while the 75 kDa signal is more prominent in females (Figure 1D, lanes 7-8). In M. domestica four major phophorylated SR proteins were detected with comparable levels in both sexes (Figure 1E, Figure lanes 7-8). In two immunoblots with samples isolated from two different biological replicates of adult C. capitata flies, we observed that phosphorylated polypeptides were highly enriched only in males (Figure 1, lanes 7 of blots in F and G). Two of the six major SR antigens detected (~75 kDa and ~30 kDa) can also be seen at much lower levels in the female samples in one of the 2 blots (Figure 1, blot in F, lanes 8). As only 0.04% of cell proteins are SR proteins [39] and the mAb104 can detect them only by Mg++ enrichment, we propose that the male-specific C. capitata SR antigens correspond most likely to phosphorylated SR proteins expressed in most of the fly tissues rather then small tissues (such as for example the male germ line).

Bottom Line: In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx).To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly).We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.

Show MeSH