asmid TKS-BP2, digested with a restriction enzyme, and inserted into pcDNA3-HANUP88. To generate pcDNA3-HA-NUP88-IR, the IR region was excised from pEGFPC3BP2IR with EcoRI and BamHI and inserted into pcDNA3-HA, and the NheIXbaI fragment containing the IR was further excised and reinserted into the XbaI site of pcDNA3-HA-NUP88. RT-PCR Total RNA was isolated using Isogen. For cDNA synthesis, 1 g of total RNA was reverse transcribed with a High Capacity cDNA Reverse Transcription Kit. Quantitative PCR of the target cDNAs was performed using Power SYBR Green PCR Master Mix. Each experiment was performed at least three times. The fold relative enrichment was quantified, with normalization to actin or GAPDH. Alternative splicing can be regulated in tissue-specific and/or stage-specific manners and can be responsive to signaling cues. Regulation of alternative splicing is achieved through the interaction of trans-acting splicing regulator complexes with the global splicing machinery. Gene-specific AS is regulated by differential expression, degradation, phosphorylation and intracellular localization of splicing factors. Each splicing factor can have antagonistic effects on the alternative splicing outputs on few specific genes or on coordinated group of genes. Serine/arginine-rich proteins are splicing factors involved in the regulation of several developmental processes, including sex determination in Insects, which share a RRM-type RNA binding domain and are rich in serine/arginine residues. Phosphorylation of the Correspondence: [email protected]; [email protected]. cn 1 Department of Biology, University of Naples Federico II, 80134, Naples, Italy 3 State Key Laboratory of Agricultural Microbiology and Institute of Urban and Horticultural Pests, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Full list of author information is available at the end of the article serine/arginine repeats provokes a structural change that alters the functional state of these splicing regulators, thereby affecting developmental choices. Protein phosphorylation plays an essential role in the regulation of sex-specific splicing in Drosophila. The LAMMER kinase DOA is responsible for phosphorylating the SR protein RBP1 and most likely other two splicing factors: TRA-2 and the female-specific TRA, which promote female-specific splicing of doublesex. DOA phosphorylates also a MedChemExpress Sodium laureth sulfate second SR major protein, DX16, which is the Drosophila ortholog of the human SFRS7 9G8 SR protein. In the Mediterranean fruit fly C. capitata and in the housefly M. domestica, the mechanism of sex determination of Drosophila is partially conserved. In these species, TRA and TRA-2, which are related to SR proteins, function as splicing regulators of doublesex and fruitless pre-mRNAs, promoting their female-specific expression and, ultimately, female sexual differentiation. Orthologs of the Drosophila tra gene in different insect orders appear to be ON/OFF 2014 Saccone et al.; licensee BioMed Central Ltd. This is an Open 
Access article distributed under the terms of the Creative Commons Attribution License, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19794399 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saccone et al. BMC Genetics 2014, 15:S6 http://www.biomedcentral.com/1471-2156/15/S2/S6 Page 2 of 6 master switch genes for sex determination and are female-specifically expressed throug