THE ORGANIZATION OF RIBOSOMAL-RNA PROCESSING CORRELATES WITH THE DISTRIBUTION OF NUCLEOLAR SNRNAS

We have analyzed the organization of pre-rRNA processing by confocal m icroscopy in pea root cell nucleoli using a variety of probes for fluo rescence in situ hybridization and immunofluorescence. Our results sho w that transcript processing within the nucleolus is spatially highly organized, Probes to the 5' external transcribed spacer (ETS) and firs t internal transcribed spacer (ITS1) showed that the excision of the E TS occurred in a sub-region of the dense fibrillar component (DFC), wh ereas the excision of ITS1 occurred in the surrounding region, broadly corresponding to the granular component, In situ labelling with probe s to the snoRNAs U3 and U14, and immunofluorescence labelling with ant ibodies to fibrillarin and SSB1 showed a high degree of coincidence wi th the ETS pattern, confirming that ETS cleavage and 18 S rRNA product ion occur in the DFC. ETS, U14, fibrillarin and SSB1 showed a fine sub structure within the DFC comprising closely packed small foci, whereas U3 appeared more diffuse throughout the DFC, A third snoRNA, 7-2/MRP, was localised to the region surrounding the ETS, in agreement with it s suggested role in ITS1 cleavage, All three snoRNAs were also frequen tly observed in numerous small foci in the nucleolar vacuoles, but non e was detectable in coiled bodies, Antibodies to fibrillarin and SSB1 labelled coiled bodies strongly, though neither protein was detected i n the nucleolar vacuoles, During mitosis, all the components analyzed, including pre-rRNA, were dispersed through the cell at metaphase, the n became concentrated around the periphery of all the chromosomes at a naphase, before being localized to the developing nucleoli at late tel ophase, Pre-rRNA (ETS and ITS1 probes), U3 and U14 were also concentra ted into small bodies, presumed to be pre-nucleolar bodies at anaphase .