Intron-independent association of splicing factors with active genes

The cell nucleus is organized as discrete domains, often associated with sp ecific events involved in chromosome organization, replication, and gene ex pression. We have examined the spatial and functional relationship between the sites of heal shock gene transcription and the speckles enriched in spl icing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90 alpha and hsp70 ge nes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription: additionally the hsp90 alpha gene contains 1 0 introns whereas the hsp70 gene is intronless, At 37 degrees C, only 30% o f hsp90 alpha transcription sites are associated with speckles whereas litt le association is detected with the hsp70 gene, whose constitutive expressi on is undetectable relative to the hsp90 alpha gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidi ne, transcription at the hsp90 alpha and hsp70 gene loci is strongly induce d, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the prese nce of intervening sequences at specific gene loci and the association with splicing factor-rich regions and suggest that subnuclear structures contai ning splicing factors are associated with sites of transcription.