Inefficient processing impairs release of RNA from the site of transcription.
We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human beta-globin genes that are either normal or defective in splicing or 3' end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, alpha-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human beta-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3' end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.
D000546:Amanitins / Q000494:pharmacology; D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D005914:Globins / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D009154:Mutation; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics; D014158:Transcription, Genetic