Early stages of pre-rRNA formation within the nucleolar ultrastructure of mouse cells studied by in situ hybridization with a 5'ETS leader probe

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5'-terminal region of the 5' external transcribed spacer (5'ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5'ETS leader region. To gain further insight into the intranucleolar localization of early stages of pre-ribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells. In control cells, 5'ETS leader rRNA was detected at the border of the fibrillar centers and over the dense fibrillar component, in agreement with previous data suggesting that rRNA gene transcription takes place at the border of the fibrillar centers before a rapid transfer of the nascent trancript to the dense fibrillar component. Observation of cells subjected to a short treatment with low doses of AMD fully supports this conclusion, with the prematurely terminated 5'ETS leader-containing transcripts detected at the border of enlarged fibrillar centers. With prolonged periods of AMD treatment even the partial transcription of rRNA genes is blocked and fibrillar centers of typically segregated nucleoli show no positive signals with the 5'ETS leader probe. We also analyzed in parallel the intranucleolar distribution of U3 small nucleolar RNA, which is involved in 5'ETS processing, by hybridization with biotinylated antisense oligonucleotides. Distribution of U3 roughly paralleled that of 5'ETS leader rRNA in untreated cells. However, U3 RNA persisted in the dense fibrillar component of segregated nucleoli whatever the conditions of drug treatment, i.e., even after a thorough chase of the rRNA precursors from this nucleolar compartment.