Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5'-fluorouridine into nascent RNA.
In this study we have used the transcription assay with 5'-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5'-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5'-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5'-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5'-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5'-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5'-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems.
Casafont
I
I
Department of Anatomy and Cell Biology and Biomedicine Unit, CSIC, University of Cantabria, Avd. Cardenal Herrera Oria, s/n, 39011 Santander, Spain.
Navascués
J
J
Pena
E
E
Lafarga
M
M
Berciano
M T
MT
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004260:DNA Repair / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology; D022041:Euchromatin / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology; D006657:Histones / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D046529:Microscopy, Electron, Transmission; D009475:Neurons, Afferent / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology; D012668:Trigeminal Ganglion / Q000378:metabolism* / Q000648:ultrastructure; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
D003609:Dactinomycin; D022041:Euchromatin; D006657:Histones; D012333:RNA, Messenger; D014529:Uridine; C001943:5-fluorouridine; C475961:gamma-H2AX protein, mouse