Yeast Ribosome Assembly Intermediates.
Tiffany Miles, Jelena Jakovljevic, Piyanun Harnpicharnchai,
Edward Horsey, John Woolford
Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, PA 15213, USA
Although there has been much progress to characterize the pathway of
processing of pre-rRNAs, understanding the mechanisms by which ribosomal
proteins (rps) assemble with rRNA to produce mature ribosomes has lagged
behind. Genetic approaches in yeast have identified more than 70
nonribosomal proteins (non-rps) necessary for ribosome assembly. Many of
these proteins are evolutionarily conserved, suggesting that the pathway
of ribosome biogenesis is largely conserved among eukaryotes. A detailed
understanding of the functions of these factors would be facilitated by
learning the context in which they function. How many different pre-ribosomal particles are present? What are the protein and RNA
constituents of each individual pre-RNP? Do rps and non-rps assemble
into pre-rRNPs individually or as subassembly complexes? We used yeast
strains expressing TAP-tagged nucleolar proteins Nop7p, Ytm1p, Rrp1p,
Nop4p or Mrt4p to purify precursors to 60S ribosomal subunits. The
affinity-purified particles are a mixture of 90S and different 66S pre-rRNPs containing 35S, 27SA, 27SB, or 7S plus 25.5S pre-rRNAs. The
particles also contain rps and non-rps, including many not previously
implicated in ribosome biogenesis. We are using genetic and biochemical
approaches to isolate individual preribosomal particles from these
mixtures and to identify subassembly complexes. We are also
characterizing pre-rRNPs that accumulate in mutants blocked in ribosome
biogenesis.
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