Fox-2 splicing factor binds to a conserved intron motif to promote inclusion 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 heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article 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. 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.

FISH in cancer diagnosis and prognostication: from cause to course of disease.

The last 20 years have witnessed an astounding evolution of cytogenetic approaches to cancer diagnosis and prognostication. Molecular techniques and, in particular, nonisotopically-labeled nucleic acid probes and fluorescence in situ hybridization (FISH)-based techniques have replaced the costly and potentially dangerous radioactive techniques used in research and the clinical detection of genetic alterations in tumor cells. Fluorescent DNA probes also enabled the screening for very subtle chromosomal changes. Clinical laboratories now choose from a growing number of FISH-based cytogenetic tests to support physician's diagnoses of the causes and the course of a disease. Depending on the specimen, state-of-the-art FISH techniques allow the localization and scoring of 10-24 different targets and overcome previous problems associated with target colocalization and detection system bandwidth. FISH-based analyses have been applied very successfully to the analysis of single cells and have demonstrated the existence of cell clones of different chromosomal make-up within human tumors. This information provides disease-specific information to the attending physician and should enable the design of patient-specific protocols for disease intervention.