Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)(+) homologue cDNA,a gene involved in ubiquitination of SmRXR1 and SmRXR2

Drosophila (SINA) and human Seven in Absentia (SIAH-1 and SIAH-2) have been implicated in ubiquitin-mediated proteolysis of different target proteins. Using the Schistosoma mansoni nuclear receptor SmRXR2 as bait in a yeast two-hybrid system, we identified a DNA fragment that encodes part of the schistosome homologue of the Seven in Absentia protein (SmSINA). Screening of S. mansoni cDNA expression library resulted in the isolation of a cDNA containing the full-length coding region of SmSINA. SmSINA contains the characteristic structural features of other SINA proteins including a conserved N-terminal RING finger domain and a cysteine-rich C-terminus. We demonstrate that SmSINA associates with SmRXR2 and SmRXR1 both in vivo and in vitro, and define the binding domains in SmRXR2 and SmRXR1 that mediate their interaction. Schistosome SINA co-localizes with SmRXR2 and SmRXR1 in vitelline cells. In addition, we show that SmSINA stimulates the ubiquination of both SmRXR2 and SmRXR1 in vitro. Our findings suggest that SmSINA regulates ubiquitination and ubiquitin-induced degradation of schistosome nuclear receptors (RXR1 and RXR2) via the ubiquitin-proteasome pathway.     

Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation

Sex-specific alternative processing of the doublesex (dsx) pre-mRNA controls somatic sexual differentiation in Drosophila melanogaster. Processing in the female-specific pattern results from the utilization of an upstream 3'-terminal exon and requires the activities of both the transformer (tra) and transformer-2 (tra-2) genes. Use of the more downstream male-specific terminal exons does not require the activities of these genes and is thus considered the default dsx-processing pattern. Here, we used transient expression of dsx pre-mRNAs in the presence or absence of tra and tra-2 gene products in Drosophila tissue culture cells to investigate the molecular mechanism controlling this alternative RNA-processing decision. These studies reveal that female-specific processing of dsx pre-mRNA is controlled by tra and tra-2 through the positive regulation of female-specific alternative 3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a 540-nucleotide region from within the female exon is both necessary and sufficient for regulation. In addition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently of female-specific polyadenylation. Regulated polyadenylation was obtained only in the presence of splicing, suggesting that activation of female-specific exon use occurs by 3'SS activation.