Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits.
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 A(0) to A(2) 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 A(3) 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 rrp5Delta6) 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 rrp5Delta6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A(2). Instead, the 32S precursor is processed directly at site A(3), 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-A(3) 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-A(2) fragment. We also show that rrp5Delta6 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.
Vos
Harmjan R
HR
Section of Biochemistry and Molecular Biology, FEW, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. vos@few.vu.nl
Faber
Alex W
AW
de Gier
Maaike D
MD
Vos
Jan C
JC
Raué
Hendrik A
HA
D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D003513:Cycloheximide / Q000494:pharmacology; D017353:Gene Deletion; D005838:Genotype; D006921:Hygromycin B / Q000494:pharmacology; D008957:Models, Genetic; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D016601:RNA-Binding Proteins; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014158:Transcription, Genetic
D000900:Anti-Bacterial Agents; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011500:Protein Synthesis Inhibitors; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; C102025:RRP5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006921:Hygromycin B; D012313:RNA; D003513:Cycloheximide