Abstract
Rps15p, an essential ribosomal protein, was previously shown to be critical for nuclear export of small subunit pre-particles. We have designed a synthetic lethal screen in Saccharomyces cerevisiæ to identify its genetic partners and further elucidate its role during ribosomal biogenesis. Our screen revealed interactions with mutants affected at various stages during ribosome biogenesis, from early nucleolar steps to nuclear export. Mutations were identified in genes encoding proteins involved in early ribosome biogenesis steps, like the small subunit processome component Utp15p, the 90S pre-ribosome factor Slx9p and the H/ACA snoRNP core protein Nhp2p. In addition, we found a synthetic lethality with BUD23, a gene encoding a methyltransferase involved both in rRNA modification and small subunit nuclear export. Interestingly, deletion of snR36 or snR85, two H/ACA snoRNAs that direct modifications close to Rps15p's binding site on the rRNA, produces mild and opposite effects on growth in an rps15 hypomorphic background. These data uncover an unreported link between a ribosomal protein and rRNA modification machinery.
Highlights
Ribosome biogenesis in eukaryotes is a complex process that takes place, for most of it, in the nucleolus, a specialized domain of the nucleus
Screening of RPS15 genetic partners To ascribe precise functions to Rps15p and identify its partners, we designed a synthetic lethal genetic screen based on a yeast hypomorphic mutant rps15-1, thermosensitive at 37uC
The mutants were assigned to two main classes: those retaining pre-40S particles in the nucleoplasm, which could be genuine mutants in the export pathway, like Ins3, Ins5, Ins13, Ins17, and Dbud23; and mutants affected in early, nucleolar steps in ribosome biogenesis, such as Ins11, nhp2ins9, utp15ins15 and Dslx9
Summary
Ribosome biogenesis in eukaryotes is a complex process that takes place, for most of it, in the nucleolus, a specialized domain of the nucleus. The pre-rRNA is subjected to a series of nucleotide modifications, mostly ribose 39O-methylations and pseudo-urydilations These modifications are catalyzed by small nucleolar ribonucleoparticles (snoRNPs) through specific base-paring between their RNA component (snoRNA) with the surrounding of the position to be modified. These RNA processing steps are intimately coupled to the assembly of diverse proteins with the precursor RNAs, which starts as soon as transcription is initiated. These proteins include the 79 ribosomal proteins, which remain associated to the mature subunits, as well as a large number of trans-acting factors. After participating in the early 18S rRNA maturation steps, these factors are released from pre-ribosomal particles when paths to form the precursors to the 40S and the 60S ribosomal subunits separate after A2 cleavage (Fig. 1)
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