Intron-independent association of splicing factors with active genes.
The cell nucleus is organized as discrete domains, often associated with specific events involved in chromosome organization, replication, and gene expression. We have examined the spatial and functional relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90alpha and hsp70 genes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription; additionally the hsp90alpha gene contains 10 introns whereas the hsp70 gene is intronless. At 37 degrees C, only 30% of hsp90alpha transcription sites are associated with speckles whereas little association is detected with the hsp70 gene, whose constitutive expression is undetectable relative to the hsp90alpha gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidine, transcription at the hsp90alpha and hsp70 gene loci is strongly induced, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the presence of intervening sequences at specific gene loci and the association with splicing factor-rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.
Jolly
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Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA. c-jolly@nwu.edu
Vourc'h
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Robert-Nicoud
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Morimoto
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D001384:Azetidines / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics*; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007438:Introns / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*; D015533:Transcriptional Activation
D001384:Azetidines; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D002104:Cadmium; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; C082735:azetidine; D012319:RNA Polymerase II