Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms

A novel family of highly conserved antigens that induce protective immunity against Staphylococcus aureus

ABSTRACT In the human pathogen Staphylococcus aureus there exists an enormous diversity of proteins containing domains of unknown function (DUF). Here, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to S. aureus. The 18 Csa paralogs in S. aureus Newman are highly similar at the sequence level yet were found to be expressed in multiple cellular localizations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed a dynamic complex formation of Csa1A with several Csa paralogs regulated by metal ions. Interestingly, the paralogs presented various modes of interaction with Csa1A, suggesting that the proteins are involved in the same cellular process in which each paralog might contribute with a particular role. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins, confirming the uniqueness of this family. Since immunization with Csa proteins protected mice from lethal challenge with S. aureus, we propose these antigens as potential vaccine candidates