A cotranscriptional model for 3′-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor

Abstract

Cleavage of the Saccharomyces cerevisiae primary ribosomal RNA (rRNA) transcript in the 3′ external transcribed spacer (ETS) by Rnt1 p generates the 35S pre-rRNA, the earliest detectable species in the pre-rRNA processing pathway. In this study we show that Rnt1p is concentrated in a subnucleolar dot-shaped territory distinct from the nucleolar body. The 35S pre-rRNA is localized at the periphery of the Rnt1p dot, in a pattern that suggests a diffusion of the 35S pre-rRNA from the site of Rnt1p processing. When plasmid-borne versions of the rDNA are used to express rRNAs, the Rnt1p territory reorganizes around these plasmids, suggesting a close association between Rnt1p and the plasmid-borne rDNA units. Rnt1 p was found associated with the endogenous rDNA by chromatin immunoprecipitation. Deletion of functionally important Rnt1p domains result in a loss of the dot-shaped territory, showing that this subnucleolar territory corresponds to a functional site of processing. These results show that a large fraction of Rnt1p is localized at the site of transcription of the rDNA, suggesting that the cleavage of the primary pre-rRNA transcript to generate the 35S pre-rRNA is a cotranscriptional event.