Each of the five snRNAs assembles with proteins to form small nuclear ribonucleoprotein particles

h alternative splicing. Furthermore, except for Drosophila, tra is also able to regulate itself, promoting female-specific splicing of its own pre-mRNA to maintain continuous expression of TRA activity. It is still unknown, though, how the male-specific splicing of tra, which leads to truncated, non-functional TRA isoforms, is regulated by the male determining factors in C. capitata and M. domestica. Furthermore it is not yet clear if and how many downstream genes promoting sexual differentiation are also being controlled, during development and in different tissues, by sex-specific alternative splicing in Drosophila and other insect species. To investigate if sex-specific SR phosphorylation takes place in adult dipteran flies, we used a commercially available mouse monoclonal antibody, which surprisingly recognizes a highly conserved phosphoepitope shared by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19796427 several major SR proteins in metazoans, including vertebrates. The SR family comprises at least 7 distinct major proteins in Drosophila and 9 in Homo sapiens; they all share the mAb104-phosphoepitope, which is also conserved in plants. Interestingly, this antibody cross-reacts also with Drosophila TRA and TRA-2 baculovirus-expressed recombinant proteins in HeLa extracts, possibly owing to their phosphorylated SR domains promoted by human cell kinases. We searched for sex-specific differences in phosphorylation of major SR splicing factors at adult stages of flies in the three distantly related dipteran species mentioned above, which are phylogenetically distant 130 million of years, with C. capitata and Drosophila more closely related. Results and discussion 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. In loading and protein transfer . 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 IMR-1 biological activity similar to those previously observed in Drosophila Kc cell line. However, the relative amounts appear different in males and females. For instance, the 95 kDa signal is significantly stronger in males, while the 75 kDa signal is more prominent in females. In M. domestica four major phophorylated SR proteins were detected with comparable levels in both sexes. 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. Two of the six major SR antigens detected can also be seen at much lower levels in the female samples in one of the 2 blots. As only 0.04% of cell proteins are SR proteins 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. When samples were treated with a phosphatase prior to immunoblotting, four of the six bands detected in male extracts disappeared. The ones that persisted even after phosphatase treatment could also be detected in the female sample. These signals probably result from mAb104 epitopes which were resistant to phosphatase treatment. This data suggests that the