Regulation of 60S ribosome biogenesis in the yeast Saccharomyces cerevisiae

Abstract

The biosynthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60S ribosomal subunits because it is essential for the processing of 35S pre-rRNA to form the mature 25S and 5.8S rRNAs. The protein is phosphorylated both in yeast and mammalian cells.;In the present work, we have carried out molecular genetic and biochemical analyses to understand the role of phosphorylation in the function of Tif6p. We show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. The site of Tif6p phosphorylation was identified to be Ser-174 (major site) by mass spectrometric analysis. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to the 66S pre-ribosomal particles that also contain bound Tif6p suggesting that phosphorylation of Tif6p may occur on the pre-66S particles in the nucleus. Furthermore, failure to phosphorylate Tif6p either by depletion of Hrr25p in a conditional yeast mutant, or by expressing unphosphorylatable Ala-mutant form of Tif6p, strongly inhibits the efficient processing of pre-rRNAs to form the mature 25S rRNA. These results, along with our previous observations that phosphorylatable serine-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60S ribosomal subunits in yeast cells.;Additionally, Tif6p and its associated proteins and RNAs were purified by using the tandem affinity purification (TAP) technique from extracts of yeast cells, expressing C-terminally TAP-tagged Tif6p. Analysis of the RNA that co-purified with Tif6p-TAP shows that Tif6p is predominantly associated with the two late 66S pre-ribosomal particles in the nucleus and not with mature 60S ribosomal particles in the cytoplasm. This observation was further confirmed by showing that under steady state growth conditions of yeast cells expressing GFP-tagged Tif6p, the protein is localized primarily in the nucleolus and not in the cytoplasm.;The implications of our work in 60S ribosome biogenesis are discussed.