Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

“…The approach that we have employed uses natural cellular mechanisms to introduce nucleotide analogs into intact cells+ We show that FuGene-6, in addition to functioning as a transfectant for plasmids, efficiently and rapidly mediates the uptake of the halogenated base analog BrUTP into cultured cells+ Although other lipid transfection reagents have been successfully used as vectors for the uptake and incorporation of BrUTP into growing cells (Haukenes et al+, 1997;Kanestrom et al+, 1998), we demonstrate that lipofection with BrUTP can be a useful tool for studying transport of newly labeled rRNAs in intact cells either within the nucleus or from the nucleus to the cytoplasm+ In summary, this nondestructive method presents several decisive advantages which permits (1) the complementary ultrastructural and optical investigations of the sites in which rRNA elongation and processing take place by using the same labeling conditions; (2) a more resolved ultrastructural identification of the nucleolar components containing the BrUTP-labeled RNAs, compared to previous autoradiographic studies (Fakan, 1978; (3) the detailed analysis of the volumic organization of the nucleolar components containing the BrUTP-labeled RNAs and of their modification along time by using confocal microscopy, three-dimensional reconstruction, and visualiza- Our ultrastructural data demonstrate that ribosome biogenesis starts in the FC and in the inner part of the DFC, then migrates rapidly in the outer part of the DFC and in the GC+ The three-dimensional reconstruction and visualization, by giving a global view, clearly indicate that the transport of rRNAs within the nucleolus occurs neither randomly nor along tracks as for some mRNA (Lawrence et al+, 1989;Smith et al+, 1999)+ On the contrary, we demonstrate that transport of rRNA follows a volumic, well-defined pathway from the inner part of the nucleolus toward its periphery+ This migration can be summarized by four typical labeling patterns, which could correspond to four main functional domains+…”

Intranuclear foci containing low abundance herpes simplex virus latency-associated transcripts visualized by non-isotopic in situ hybridization

“…Should processed minor LAT RNAs be transported to the cytoplasm it is likely that they are present at very low concentrations, making them undetectable with our current ISH protocol. This situation would be analogous to that of the EBV RNAs encoding viral nuclear antigens, which are readily detectable within nuclei during latent infection but which cannot be detected within the cytoplasm of these cells (Lawrence et al, 1989;Xing & Lawrence, 1991).…”

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

“…Not only a number of nuclear antigens, but also RNA transcripts have been found to accumulate in the nucleus (Lawrence et al 1989;Raap et al 1991). Following visualization by in situ hybridization, these RNA concentrations can be seen as punctate spots or track-like objects.…”