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

AUTOR(ES)

Vos, Harmjan R.

FONTE

American Society for Microbiology

RESUMO

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 rrp5Δ6) 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 rrp5Δ6 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 rrp5Δ6 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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