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Eukaryotic Cell, December 2004, p. 1504-1512, Vol. 3, No. 6
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.6.1504-1512.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Deletion of the Three Distal S1 Motifs of Saccharomyces cerevisiae Rrp5p Abolishes Pre-rRNA Processing at Site A₂ without Reducing the Production of Functional 40S Subunits

Section of Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Exact Sciences and Institute of Molecular Biological Science, BioCenter Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands

Received 3 May 2004/ Accepted 17 August 2004

Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A₀ to A₂ and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A₃ and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp56) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in the rrp56 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A₂. Instead, the 32S precursor is processed directly at site A₃, producing exclusively 21S rather than 20S pre-rRNA. This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis. Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation. The resulting D-A₃ fragment, however, is degraded by both 5'3' and 3'5' exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5'3' exonucleolytic digestion of the D-A₂ fragment. We also show that rrp56 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal.

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