Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in <i>Saccharomyces cerevisiae</i>

Dembowski, Jill A.; Ramesh, Madhumitha; McManus, C. Joel; Woolford, John L.

doi:10.1261/rna.041194.113

Cited by 26 publications

(34 citation statements)

References 48 publications

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“…Interestingly, we observe increased modification of 2 nt in the Cic1 interaction site, which may indicate a change in the binding of Cic1. Moreover, we observe changes in reactivity at a common subset of nucleotides that would be consistent with a shift from ring structure to hairpin structure of ITS2 (Granneman et al 2011;Babiano et al 2013;Dembowski et al 2013). Thus, in the absence of the N-terminal extension of L8, the binding of some assembly factors that interact with ITS2 are distorted, which may cause ITS2 to form a structure that inhibits its processing after C 2 cleavage.…”

Section: Resultssupporting

confidence: 57%

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

Tutuncuoglu

Jakovljevic

et al. 2016

RNA

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Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo-that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling.

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Section: Resultssupporting

confidence: 57%

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

Tutuncuoglu

Jakovljevic

et al. 2016

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“…Taken together, these results suggest that removal of ITS1 and ITS2 requires cou-pling between these sequences that may be located far apart within preribosomes. Consistent with this, depletion of proteins that bind to ITS2 (Cic1, Nop15, and Rlp7) causes ITS2 to misfold but inhibit removal of ITS1 (26,(30)(31)(32)(33). By binding to the ITS2-proximal stem, the Pwp1 subcomplex appears to be located in the preribosome at a position to enable long-range communication between these two spacers (Fig.…”

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confidence: 59%

“…Sequences of rRNA cross-linked to Nop12, Nop7, and Erb1 are shown in red, green, and magenta, respectively (26). ITS2 is represented by the dashed line to indicate the approximate locations of Cic1, Nop15, and Rlp7 (26,30).…”

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confidence: 99%

Ribosome Assembly Factors Pwp1 and Nop12 Are Important for Folding of 5.8S rRNA during Ribosome Biogenesis in Saccharomyces cerevisiae

Talkish

Campbell

Sahasranaman

et al. 2014

Molecular and Cellular Biology

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Previous work from our lab suggests that a group of interdependent assembly factors (A 3 factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A 3 factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA 3 pre-rRNA processing, even though the A 3 factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A 3 factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures. Ribosome assembly is a highly conserved and dynamic process driven by the cooperative transcription, folding, modification, and processing of rRNAs and stable binding of ribosomal proteins (r-proteins) (1, 2). In Saccharomyces cerevisiae, this process begins in the nucleolus with cotranscriptional assembly of early precursor particles. As transcription proceeds, the nascent pre-rRNA is cleaved, separating maturation of early 40S and 60S precursors. A series of endo-and exonucleolytic cleavages remove internal and external transcribed spacer sequences from prerRNAs, as the assembling ribosome moves from the nucleolus to the nucleoplasm and is eventually exported to the cytoplasm ( Fig. 1A and B).Assembly of yeast ribosomes requires ϳ180 trans-acting proteins termed assembly factors (AFs). The majority of these proteins are conserved across eukaryotes, are essential for cell growth, and are thought to function as scaffolding proteins, RNA chaperones, energy-consuming nucleoside triphosphatases (NTPases), nucleases, or posttranslational modifiers (3-5). Purification of ribosome assembly intermediates allowed the identification of most AFs, and initial characterizations have shown in which steps of pre-rRNA processing many of these factors function. However, to begin to understand the function of these proteins on a mechanistic level, one must address the following: the timing of association of these proteins with preribosomes; how they are recruited to preribosomes; their requirement for the stable binding of other proteins; and their role in folding rRNA.Numerous studies have shown that subsets of AFs can be isolated as subcomplexes (6-23). Proteins within a subcomplex are often required for the same steps of pre-rRNA processing, suggesting that they function together during ribosome bi...

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“…Furthermore, it has been previously pointed out that eukaryotic RNA elements and eukaryotic r-proteins and their extensions localize together on the solvent-exposed side of the ribosome and make numerous contacts with each other (Ben-Shem et al 2011). Several eukaryote-specific assembly factors such as Rlp7, Arx1, Rrp5, and Nop7 bind to ES (Granneman et al 2011;Bradatsch et al 2012;Babiano et al 2013;Dembowski et al 2013;Lebaron et al 2013). Combined with our observation about similar functions in ribosome assembly for eukaryote-specific ES31Δ L and the eukaryote-specific extension of L8, these lines of evidence raise the possibility that eukaryote-specific protein-RNA elements may have coevolved, and perform similar functions in the cell.…”

Section: Viable Es Mutants Can Synthesize Mature 25s Rrnamentioning

confidence: 99%

Eukaryote-specific rRNA expansion segments function in ribosome biogenesis

Ramesh

Woolford

2016

RNA

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The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis.

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Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Cited by 26 publications

References 48 publications

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

Ribosome Assembly Factors Pwp1 and Nop12 Are Important for Folding of 5.8S rRNA during Ribosome Biogenesis in Saccharomyces cerevisiae

Eukaryote-specific rRNA expansion segments function in ribosome biogenesis

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