The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.
The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are necessary for 3'-terminal processing of polyadenylated mRNAs. To study the distribution of 3' cleavage factors in the nuclei of human T24 cells, monoclonal antibodies against the CstF 64 kDa subunit and against the CPSF 100 kDa subunit were used for immunofluorescent labelling. CstF 64 kDa and CPSF 100 kDa were distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1-4 bright foci. Double immunofluorescence labelling experiments revealed that the foci either overlapped with, or resided next to, a coiled body. Inhibition of transcription with alpha-amanitin or 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole (DRB) resulted in the complete co-localization of coiled bodies and foci containing 3' cleavage factors. Electron microscopy on immunogold double-labelled cells revealed that the foci represent compact spherical fibrous structures, we named 'cleavage bodies', intimately associated with coiled bodies. We found that approximately 20% of the cleavage bodies contained a high concentration of newly synthesized RNA, whereas coiled bodies were devoid of nascent RNA. Our results suggest that the cleavage bodies that contain RNA are those that are adjacent to a coiled body. These findings reveal a dynamic and transcription-dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific transcripts.
Schul
W
W
E.C. Slater Institute, Biocentrum, University of Amsterdam, The Netherlands.
Groenhout
B
B
Koberna
K
K
Takagaki
Y
Y
Jenny
A
A
Manders
E M
EM
Raska
I
I
van Driel
R
R
de Jong
L
L
D000546:Amanitins / Q000494:pharmacology; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D039221:mRNA Cleavage and Polyadenylation Factors
D000546:Amanitins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D012319:RNA Polymerase II