Identification of cis-acting elements involved in 3′-end formation of Saccharomyces cerevisiae 18S rRNA

Beekvelt, C.A. van; Jeeninga, Rienk E.; Riet, J. van’t; Venema, Jaap; Raué, Hendrik A.

doi:10.1017/s1355838201010196

Cited by 12 publications

(12 citation statements)

References 38 publications

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“…This finding is consistent with the observation that the ability to form a duplex at site D is not conserved in eukaryotes (40,41). Furthermore, mutations that would disrupt a duplex have no effect on rRNA maturation or stability (42).…”

Section: Discussionmentioning

confidence: 99%

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

Lamanna¹,

Karbstein

2009

Proc. Natl. Acad. Sci. U.S.A.

129

157

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Ribosome assembly is a hierarchical process that involves pre-rRNA folding, modification, and cleavage and assembly of ribosomal proteins. In eukaryotes, this process requires a macromolecular complex comprising over 200 proteins and RNAs. Whereas the rRNA modification machinery is well-characterized, rRNA cleavage to release mature rRNAs is poorly understood, and in yeast, only 2 of 8 endonucleases have been identified. The essential and conserved ribosome assembly factor Nob1 has been suggested to be the endonuclease responsible for generating the mature 3-end of 18S rRNA by cleaving at site D. Here we provide evidence that recombinant Nob1 forms a tetramer that binds directly to pre-rRNA analogs containing cleavage site D. Analysis of Nob1's affinity to a series of RNA truncations, as well as Nob1-dependent protections of pre-rRNA in vitro and in vivo demonstrate that Nob1's binding site centers around the 3-end of 18S rRNA, where our data also locate Nob1's suggested active site. Thus, Nob1 is poised for cleavage at the 3-end of 18S rRNA. Together with prior data, these results strongly implicate Nob1 in cleavage at site D. In addition, our data provide evidence that the cleavage site at the 3-end of 18S rRNA is single-stranded and not part of a duplex as commonly depicted. Using these results, we have built a model for Nob1's interaction with preribosomes.ribosome assembly ͉ rRNA cleavage R ibosomes catalyze protein synthesis in all cells. As such, their function and biogenesis have been extensively studied. Classic work in bacteria has characterized the order in which ribosomal proteins (r-proteins) assemble onto ribosomal RNA (rRNA) (1-3). Furthermore, by studying subdomains of the small subunit, it was shown that ordered assembly originates from rRNA conformational changes induced by binding of a subset of r-proteins (4-9). In vivo studies of yeast r-protein assembly suggest rough conservation of the order of r-protein assembly with ribosomes: If the bacterial homolog is a primary binder, depletion leads to an early ribosome assembly defect, while depletion of tertiary binder homologs often results in later assembly defects (10, 11). Despite these similarities ribosome assembly in vivo differs between prokaryotes and eukaryotes. In yeast, over 200 protein and RNA cofactors are required for ribosome assembly (12). Generally, these factors are conserved in eukaryotes but, with few exceptions, are absent in bacteria (13). These assembly factors orchestrate modification and cleavage of the initial 35S precursor rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs, folding of the rRNA, and binding of ribosomal proteins and 5S rRNA. Whereas rRNA modification is well-studied (14, 15), cleavage of the pre-rRNA to release mature rRNAs is poorly understood. Although the order of processing steps has been elucidated, the identity of 6 of 8 endonucleases remains unknown.Assembly of the small 40S ribosomal subunit in yeast occurs in 2 phases: An early nucleolar stage, comprising transcription, rRNA modification, ...

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Section: Discussionmentioning

confidence: 99%

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

Lamanna¹,

Karbstein

2009

Proc. Natl. Acad. Sci. U.S.A.

129

157

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“…Effect of mutation of site D on the maturation of 21S pre-rRNA. Strain YJV207 expressing Rrp5⌬6p was transformed with either plasmid pT, encoding a tagged, wild-type rDNA unit, or pTM3, encoding a tagged, mutant rDNA unit that carries a 4-nt substitution mutation across site D which inhibits cleavage at this site (46). Total RNA was isolated from both types of transformants (lanes 1 and 3 or lanes 2 and 4, respectively) and separated on an 8% polyacrylamide gel.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmids pTRP1-ProtA::rrp5 and pTRP1-ProtA::rrp5⌬6 were described by Eppens et al (18). Mutant ribosomal DNA (rDNA) plasmids pT and pTM3 were described by Van Beekvelt et al (46). Yeast transformation was performed as described by Gietz et al (24).…”

Section: Methodsmentioning

confidence: 99%

“…1A). cis-acting substitution mutation of the 4-nt region spanning site D (mutation M3), which completely inhibits this cleavage (46). The rrp5⌬6 strain was transformed with an rDNA plasmid (pTM3) that contains mutation M3 as well as a neutral tag in the 18S rRNA sequence (50), allowing discrimination between plasmid-derived and endogenous pre-rRNAs or rRNAs.…”

Section: Vol 3 2004mentioning

confidence: 99%

“…CELL M3 mutation (Fig. 7), which inhibits cleavage at site D (46), as well as the detection of a D-A 3 fragment in rrp5⌬6 mutant cells (Fig. 8).…”

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confidence: 93%

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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

et al. 2004

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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 rrp5⌬6) 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 rrp5⌬6 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 533 and 335 exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 533 exonucleolytic digestion of the D-A 2 fragment. We also show that rrp5⌬6 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.Ribosome biogenesis in eukaryotes largely takes place in the nucleolus, a specialized compartment of the nucleus, and proceeds via a complex series of steps, including transcription, modification, and processing of the pre-rRNA, integrated with the ordered assembly of various precursor species with ribosomal proteins.Pre-rRNA transcription involves multiple copies of two types of transcriptional units, one encoding 5S rRNA and the other containing the genes for 18S, 5.8S, and 25S/28S rRNAs. The latter species therefore are transcribed as a single large precursor, which contains external transcribed spacers at either end (5Ј-ETS and 3Ј-ETS) as well as two internal transcribed spacers (ITS1 and ITS2) separating the mature rRNA sequences (Fig. 1A). This primary transcript is modified extensively by ribose methylation as well as pseudouridylation (14, 15), followed by removal of the spacers in a series of endonucleolytic and exonucleolytic processing reactions.Eukaryotic pre-rRNA maturation has been characterized in most detail in the yeast Saccharomyces cerevisiae (reviewed in references 20, 28, 38, and 52) (Fig. 1B), where the first detectable pre-rRNA molecule is the 35S species, generated through endonucleolytic cleav...

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Ribosome, Chemistry of

Karbstein

2008

Wiley Encyclopedia of Chemical Biology

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Ribosomes are the most complex, ancient, and important RNA‐protein complexes present in every cell. A large body of previous data shows that their function is carefully regulated and quality controlled, which leads to a remarkably low rate of amino‐acid misincorporation. A prerequisite to this exquisitely accurate function is correctly assembled ribosomes. Here, I consider mechanisms that may help in regulating ribosome assembly and in ensuring that ribosomes are faithfully assembled. Evidence for the existence of these mechanisms is then presented based on data in the literature. Future experiments will be required to test the importance of these events in quality control.

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Identification of cis-acting elements involved in 3′-end formation of Saccharomyces cerevisiae 18S rRNA

Cited by 12 publications

References 38 publications

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

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

Ribosome, Chemistry of

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Identification of cis-acting elements involved in 3′-end formation of Saccharomyces cerevisiae 18S rRNA

Cited by 12 publications

References 38 publications

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain

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

Ribosome, Chemistry of

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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