Interactions among Ytm1, Erb1, and Nop7 Required for Assembly of the Nop7-Subcomplex in Yeast Preribosomes

In Saccharomyces cerevisiae, more than 180 assembly factors associate with preribosomes to enable folding of pre-rRNA, recruitment of ribosomal proteins, and processing of pre-rRNAs to produce mature ribosomes. To examine the molecular architecture of preribosomes and to connect this structure to functions of each assembly factor, assembly subcomplexes have been purified from preribosomal particles. The Nop7-subcomplex contains three assembly factors: Nop7, Erb1, and Ytm1, each of which is necessary for conversion of 27SA3 pre-rRNA to 27SBS pre-rRNA. However, interactions among these three proteins and mechanisms of their recruitment and function in pre-rRNPs are poorly understood. Here we show that Ytm1, Erb1, and Nop7 assemble into preribosomes in an interdependent manner. We identified which domains within Ytm1, Erb1, and Nop7 are necessary for their interaction with each other and are sufficient for recruitment of each protein into preribosomes. Dominant negative effects on growth and ribosome biogenesis caused by overexpressing truncated Ytm1, Erb1, or Nop7 constructs, and recessive phenotypes of the truncated proteins revealed not only interaction domains but also other domains potentially important for each protein to function in ribosome biogenesis. Our data suggest a model for the architecture of the Nop7-subcomplex and provide potential functions of domains of each protein.

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Ubiquitin and Ubiquitin-Like Proteins and Domains in Ribosome Production and Function: Chance or Necessity?

International Journal of Molecular Sciences ◽

10.3390/ijms22094359 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4359

Author(s):

Sara Martín-Villanueva ◽

Gabriel Gutiérrez ◽

Dieter Kressler ◽

Jesús de la Cruz

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Cellular Processes ◽

Substrate Protein ◽

Precursor Proteins ◽

Assembly Factors ◽

Ubiquitin Moiety ◽

And Function

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.

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Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1

Molecular and Cellular Biology ◽

10.1128/mcb.00668-07 ◽

2007 ◽

Vol 27 (19) ◽

pp. 6581-6592 ◽

Cited By ~ 66

Author(s):

Brigitte Pertschy ◽

Cosmin Saveanu ◽

Gertrude Zisser ◽

Alice Lebreton ◽

Martin Tengg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Export ◽

Ribosome Biogenesis ◽

Dominant Negative ◽

Cytoplasmic Localization ◽

Atpase Domain ◽

Aaa Atpase ◽

Cytoplasmic Accumulation ◽

Functional Inactivation ◽

Aaa Protein

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

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Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness

eLife ◽

10.7554/elife.43002 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 25

Author(s):

Blake W Tye ◽

Nicoletta Commins ◽

Lillia V Ryazanova ◽

Martin Wühr ◽

Michael Springer ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Maximal Growth ◽

Proliferating Cells ◽

Cellular Processes ◽

Direct Contribution ◽

The Face ◽

Cellular Phenotypes

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

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Expanding Role of Ubiquitin in Translational Control

International Journal of Molecular Sciences ◽

10.3390/ijms21031151 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1151 ◽

Cited By ~ 3

Author(s):

Shannon E. Dougherty ◽

Austin O. Maduka ◽

Toshifumi Inada ◽

Gustavo M. Silva

Keyword(s):

Translational Control ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Protein Quality ◽

Rna Binding Proteins ◽

Ubiquitin Chain ◽

Molecular Aspects ◽

And Function

The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.

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A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

The Journal of Cell Biology ◽

10.1083/jcb.139.7.1655 ◽

1997 ◽

Vol 139 (7) ◽

pp. 1655-1661 ◽

Cited By ~ 68

Author(s):

Jonathan S. Rosenblum ◽

Lucy F. Pemberton ◽

Günter Blobel

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Import ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Sequence Similarity ◽

Wild Type ◽

Major Pathway ◽

Trna Processing ◽

Transport Factors ◽

Limited Sequence

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.

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Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors

Eukaryotic Cell ◽

10.1128/ec.00172-12 ◽

2012 ◽

Vol 11 (10) ◽

pp. 1276-1288 ◽

Cited By ~ 10

Author(s):

Austin U. Gehret ◽

Sara M. Connelly ◽

Mark E. Dumont

Keyword(s):

Saccharomyces Cerevisiae ◽

G Protein ◽

Dominant Negative ◽

Negative Effects ◽

Content Type ◽

Functional Interactions ◽

Wide Range ◽

Physical Interactions ◽

Mutant Receptors ◽

G Protein Coupled

ABSTRACT The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MAT a haploid cells of the yeast Saccharomyces cerevisiae . Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-negative effects of mutant receptors persist over a wide range of ratios of the abundances of G protein to receptor and that such effects are not blocked by covalent fusion of G protein α subunits to normal receptors. In addition, we detected dominant effects of mutant C-terminally truncated receptors, which had not been previously reported to act in a dominant manner. Furthermore, coexpression of C-terminally truncated receptors with constitutively active mutant receptors results in enhancement of constitutive signaling. Together with previous evidence for oligomerization of Ste2p receptors, these results are consistent with the idea that functional interactions between coexpressed receptors arise from physical interactions between them rather than from competition for limiting downstream components, such as G proteins.

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The DEAH-box RNA helicase Dhr1 contains a remarkable carboxyl terminal domain essential for small ribosomal subunit biogenesis

Nucleic Acids Research ◽

10.1093/nar/gkz529 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7548-7563 ◽

Cited By ~ 3

Author(s):

Amlan Roychowdhury ◽

Clément Joret ◽

Gabrielle Bourgeois ◽

Valérie Heurgué-Hamard ◽

Denis L J Lafontaine ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Rna Helicases ◽

Range Interaction ◽

Small Ribosomal Subunit ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Assembly Factors ◽

Carboxyl Terminal

Abstract Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases.

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Ribosome biogenesis factor Ltv1 chaperones the assembly of the small subunit head

The Journal of Cell Biology ◽

10.1083/jcb.201804163 ◽

2018 ◽

Vol 217 (12) ◽

pp. 4141-4154 ◽

Cited By ~ 11

Author(s):

Jason C. Collins ◽

Homa Ghalei ◽

Joanne R. Doherty ◽

Haina Huang ◽

Rebecca N. Culver ◽

...

Keyword(s):

Cancer Cells ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Breast Cancer Cells ◽

Ribosomal Subunit ◽

Small Subunit ◽

Yeast Genetics ◽

Structure Probing ◽

Rna Quality Control ◽

Assembly Factor

The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.

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Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10

Cells ◽

10.3390/cells8091035 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1035 ◽

Cited By ~ 8

Author(s):

Sophie Sleiman ◽

Francois Dragon

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

40S Ribosomal Subunit ◽

Functional Features ◽

And Function ◽

Progeria Syndrome ◽

Hutchinson Gilford Progeria Syndrome ◽

Nucleolar Rna

Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.

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Ribosome Dysfunction and Ineffective Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.sci-11.sci-11 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-11-SCI-11

Author(s):

Benjamin L. Ebert

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Bone Marrow Failure ◽

Ribosomal Genes ◽

Limb Bud ◽

Refractory Anemia ◽

Diamond Blackfan Anemia ◽

Rare Congenital Disorder ◽

Human Disorders ◽

And Function

Abstract Abstract SCI-11 The 5q- syndrome and Diamond Blackfan Anemia are related on a molecular level by ribosome dysfunction. The 5q- syndrome is a distinct subtype of myelodysplastic syndrome (MDS) associated with isolated, interstitial deletions of the long arm of Chromosome 5. Diamond Blackfan Anemia is a rare congenital disorder associated with bone marrow failure, craniofacial abnormalities, and limb bud defects. The hematologic phenotype of both diseases includes a severe refractory anemia, macrocytosis, and a deficiency of erythroid precursors. Recent evidence indicates that this erythroid defect is caused by RPS14 deletion in the 5q- syndrome, and by mutation of RPS19 or other ribosomal genes in at least 50% of patients with Diamond Blackfan Anemia. In both diseases, deletion or mutation of one allele of a ribosomal protein leads to defects in pre-rRNA processing and defective production of mature ribosomes. In murine and zebrafish models, haploinsufficiency for ribosomal genes phenocopies the erythroid failure characteristic of the human disorders. While the mechanistic consequences of ribosomal dysfunction have not been fully elucidated, the p53 pathway appears to play a central role. MDM2, an E3 ubiquitin ligase that promotes the degradation of p53, binds to several ribosomal proteins including RPL11. Deficiency of RPS6, and perhaps other ribosomal proteins, causes an accumulation of free RPL11, that binds to MDM2, preventing MDM2 from interacting with p53, thereby leading to an accumulation of p53. Several other genes that are mutated in hematologic disorders are involved in ribosome biogenesis and function, including SBDS, mutated in Shwachman Diamond syndrome; DKC1, mutated in some cases of dyskaratosis congenita; and NPM1, mutated in acute myeloid leukemia and deleted in some cases of MDS. The manner in which each of these genes disrupt ribosome function and cause distinct clinical phenotypes is currently under investigation. Disclosures Ebert: GlaxoSmithKline: Research Funding.

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