Abstract
Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud.
Highlights
The synthesis of ribosomes starts with the assembly of the precursors of the 40S and 60S subunits in the nucleolus and, subsequently, progress along independent pathways that take place both in the nucleoplasm and the cytoplasm
Consistent with earlier studies (Schäfer et al 2003; Moriggi et al 2014), we found using epifluorescence microscopy analyses that a version of Tsr1 tagged at its carboxyl terminus with green fluorescent protein (GFP) localizes in the nucleolus when monitored both in asynchronously growing cells and in cells going through mitosis after synchronization in S phase with hydroxyurea (Fig. 1A,B)
We unexpectedly found the consistent accumulation during early mitosis of Tsr1-GFP in a well-defined extranucleolar site that localizes in the bud side of the nucleus in a position opposite to the nucleolus (Fig. 1B)
Summary
The synthesis of ribosomes starts with the assembly of the precursors of the 40S and 60S subunits in the nucleolus and, subsequently, progress along independent pathways that take place both in the nucleoplasm and the cytoplasm (for a scheme, see Fig. 2A). The first step of ribosome synthesis is the transcription by RNA polymerase I (Pol I) of the 35S pre-rRNA, the initial RNA precursor that contains the sequences for the mature 18S, 5.8S, and 25S rRNAs (Woolford and Baserga 2013). The pre-60S particle has to go through extensive maturation steps in the nucleoplasm that involve the engagement of more than 40 trans-acting factors (Woolford and Baserga 2013; Nerurkar et al 2015; Greber 2016; Wu et al 2016). Both the pre-40S and pre-60S particles have to undergo final structural changes and quality control mechanisms before entering the pool of functional ribosomes (Strunk et al 2011; Lebaron et al 2012; Karbstein 2013; García-Gómez et al 2014; Hector et al 2014; Turowski et al 2014)
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