scholarly journals Bud23 promotes the progression of the Small Subunit Processome to the pre-40S ribosome in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Joshua J. Black ◽  
Richa Sardana ◽  
Ezzeddine W. Elmir ◽  
Arlen W. Johnson
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Interaction Network ◽  
Small Subunit ◽  
Cellular Growth ◽  
Physical Interaction ◽  
U3 Snorna ◽  
Assembly Pathway ◽  
Genes Encoding ◽  
Ssu Processome

AbstractThe first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not well understood. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of 18S rRNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the suppressing mutations weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the GTPase Bms1 and the RNA helicase Dhr1. Bms1 is thought to drive conformational changes to promote rRNA cleavage, and we previously showed that Dhr1 is required for unwinding the U3 snoRNA. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and pre-rRNAs not cleaved at sites A1 and A2. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.Author summaryRibosomes are the molecular machines that synthesize proteins and are composed of a large and a small subunit which carry out the essential functions of polypeptide synthesis and mRNA decoding, respectively. Ribosome production is tightly linked to cellular growth as cells must produce enough ribosomes to meet their protein needs. However, ribosome assembly is a metabolically expensive pathway that must be balanced with other cellular energy needs and regulated accordingly. In eukaryotes, the small subunit (SSU) Processome is a metastable intermediate that ultimately progresses towards a mature SSU through the release of biogenesis factors. The decision to progress the SSU Processome is thought to be an early checkpoint in the SSU assembly pathway, but what drives this checkpoint is unknown. Previous studies suggest that Bud23 plays an uncharacterized role during SSU Processome progression. Here, we used a genetic approach to understand its function and found that Bud23 is connected to a network of factors that stabilize the particle. Interestingly, two of these factors are enzymes that facilitate structural rearrangements needed for progression. We conclude that Bud23 promotes the release of factors surrounding its binding site to drive rearrangements during the progression of the SSU Processome.

scholarly journals Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009215
Author(s):  
Joshua J. Black ◽  
Richa Sardana ◽  
Ezzeddine W. Elmir ◽  
Arlen W. Johnson
Keyword(s):  
Binding Site ◽  
Ribosomal Rna ◽  
Rna Processing ◽  
Interaction Network ◽  
Small Subunit ◽  
Physical Interaction ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Ribosomal Rna Processing ◽  
Ssu Processome

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.

scholarly journals The Small Subunit Processome Is Required for Cell Cycle Progression at G1

2004 ◽  
Vol 15 (11) ◽  
pp. 5038-5046 ◽  
Author(s):  
Kara A. Bernstein ◽  
Susan J. Baserga
Keyword(s):  
Cell Cycle ◽  
Ribosome Biogenesis ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Cellular Growth ◽  
Yeast Cells ◽  
G1 Phase ◽  
Relay Cell ◽  
Ssu Processome ◽  
Nucleolar Rna

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

scholarly journals Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

RNA ◽  
2021 ◽  
pp. rna.079025.121
Author(s):  
Joshua J Black ◽  
Arlen W Johnson
Keyword(s):  
Binding Site ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Assembly Line ◽  
Small Subunit ◽  
Nucleotide Hydrolysis ◽  
Ribonucleoprotein Complexes ◽  
Intermediate States ◽  
Ssu Processome

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

scholarly journals Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA

2020 ◽  
Vol 48 (7) ◽  
pp. 3848-3868 ◽  
Author(s):  
Guillaume Clerget ◽  
Valérie Bourguignon-Igel ◽  
Nathalie Marmier-Gourrier ◽  
Nicolas Rolland ◽  
Ludivine Wacheul ◽  
...  
Keyword(s):  
Protein Interaction Network ◽  
18S Rrna ◽  
Direct Interaction ◽  
Interaction Network ◽  
Specific Protein ◽  
Rrna Processing ◽  
Protein Protein Interaction ◽  
U3 Snorna ◽  
Ssu Processome ◽  
Protein Protein Interaction Network

Abstract U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5′-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

scholarly journals Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

2021 ◽  
Author(s):  
Joshua J Black ◽  
Arlen W Johnson
Keyword(s):  
Binding Site ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Assembly Line ◽  
Small Subunit ◽  
Nucleotide Hydrolysis ◽  
Ribonucleoprotein Complexes ◽  
Intermediate States ◽  
Ssu Processome

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

scholarly journals Nucleolar proteins Bfr2 and Enp2 interact with DEAD-box RNA helicase Dbp4 in two different complexes

2013 ◽  
Vol 42 (5) ◽  
pp. 3194-3206 ◽  
Author(s):  
Sahar Soltanieh ◽  
Martin Lapensée ◽  
François Dragon
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Helicase ◽  
Small Subunit ◽  
Specific Protein ◽  
18S Ribosomal Rna ◽  
Dead Box ◽  
Nucleolar Proteins ◽  
U3 Snorna ◽  
Ssu Processome

AbstractDifferent pre-ribosomal complexes are formed during ribosome biogenesis, and the composition of these complexes is highly dynamic. Dbp4, a conserved DEAD-box RNA helicase implicated in ribosome biogenesis, interacts with nucleolar proteins Bfr2 and Enp2. We show that, like Dbp4, Bfr2 and Enp2 are required for the early processing steps leading to the production of 18S ribosomal RNA. We also found that Bfr2 and Enp2 associate with the U3 small nucleolar RNA (snoRNA), the U3-specific protein Mpp10 and various pre-18S ribosomal RNA species. Thus, we propose that Bfr2, Dbp4 and Enp2 are components of the small subunit (SSU) processome, a large complex of ∼80S. Sucrose gradient sedimentation analyses indicated that Dbp4, Bfr2 and Enp2 sediment in a peak of ∼50S and in a peak of ∼80S. Bfr2, Dbp4 and Enp2 associate together in the 50S complex, which does not include the U3 snoRNA; however, they associate with U3 snoRNA in the 80S complex (SSU processome). Immunoprecipitation experiments revealed that U14 snoRNA associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the ‘50S’ complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome.

scholarly journals DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

2014 ◽  
Vol 35 (5) ◽  
pp. 816-830 ◽  
Author(s):  
Sahar Soltanieh ◽  
Yvonne N. Osheim ◽  
Krasimir Spasov ◽  
Christian Trahan ◽  
Ann L. Beyer ◽  
...  
Keyword(s):  
Rna Helicase ◽  
Sucrose Density Gradient ◽  
Small Subunit ◽  
Specific Protein ◽  
Rrna Synthesis ◽  
Catalytic Core ◽  
Dead Box ◽  
Cell Extracts ◽  
U3 Snorna ◽  
Ssu Processome

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

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