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
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
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DOI：
10.1128/ec.3.6.1504-1512.2004
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发表时间：
2004-12
期刊：
影响因子：
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通讯作者：
H. Vos;Alex W. Faber;Maaike D de Gier;J. C. Vos;H. A. Raué
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作者：
H. Vos;Alex W. Faber;Maaike D de Gier;J. C. Vos;H. A. Raué
酵母Rrp5p是两种核糖体亚基生物发生所需的少数反式作用蛋白之一，具有显著的双结构域结构。它的c端由7个四肽基序组成，其中一些基序对A0到A2位点的裂解至关重要，从而对18S rRNA的形成至关重要。另一方面，n端区域包含12个S1 rna结合基序，其中大部分是在A3位点加工所需的，从而产生5.8S rRNA的短形式。表达缺乏S1基序10至12的Rrp5p蛋白突变体（突变体rrp5Δ6）的酵母细胞具有正常的生长速度和成熟rRNA物种的野生型稳态水平，这表明这些基序与核糖体的生物发生无关。然而，在rrp5Δ6突变体中，pre-rRNA加工遵循另一种途径，不包括32S pre-rRNA在A2位点的切割。相反，32S前体直接在A3位点加工，只产生21S而不是20S前rrna。这是第一个证明21S前体是18S rRNA合成的有效底物的证据，而21S前体以前只在表现出严重生长缺陷的细胞中观察到，或者作为正常20S前体之外的次要物种。21S前体的成熟发生在与20S前rrna成熟相同的D位点核内裂解裂解。然而，产生的D-A3片段可以被5 ‘→3 ’和3 ‘→5 ’外切酶酶切降解，后者涉及外切体，而D-A2片段只能被5 ‘→3 ’外切酶切。我们还发现rrp5Δ6细胞对潮霉素B和环己亚胺都过敏，这表明，尽管它们的野生型生长速度快，但它们的核糖体或核糖体可能在结构上异常。
ABSTRACT 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.