scholarly journals Direct Link between RACK1 Function and Localization at the Ribosome In Vivo

2008 ◽  
Vol 29 (6) ◽  
pp. 1626-1634 ◽  
Author(s):  
Scott M. Coyle ◽  
Wendy V. Gilbert ◽  
Jennifer A. Doudna
Keyword(s):  
Signaling Pathways ◽  
Ribosomal Subunit ◽  
Translation Elongation ◽  
Ribosome Binding ◽  
Charged Amino Acids ◽  
40S Ribosomal Subunit ◽  
Increased Sensitivity ◽  
Mrna Binding ◽  
Ribosome Association

ABSTRACT The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-Å crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the β-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.

scholarly journals yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1384 ◽  
Author(s):  
Kerstin Schmitt ◽  
Oliver Valerius
Keyword(s):  
Quality Control ◽  
Binding Proteins ◽  
Ribosomal Subunit ◽  
Stress Factors ◽  
Head Region ◽  
Ubiquitin E3 Ligase ◽  
Mrna Binding Proteins ◽  
40S Ribosomal Subunit ◽  
Mrna Binding

Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The Saccharomyces cerevisiae protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit (hr40S) and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the hr40S. We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of hr40S localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.

scholarly journals A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

1996 ◽  
Vol 16 (10) ◽  
pp. 5328-5334 ◽  
Author(s):  
N Méthot ◽  
M S Song ◽  
N Sonenberg
Keyword(s):  
Aspartic Acid ◽  
Ribosomal Subunit ◽  
Direct Interaction ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Ribosome Binding ◽  
Initiation Factors ◽  
Self Association

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

scholarly journals The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

2020 ◽  
Author(s):  
Laura Plassart ◽  
Ramtin Shayan ◽  
Christian Montellese ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Inactive Form ◽  
Final Maturation ◽  
Translation Apparatus ◽  
Translation Accuracy ◽  
Functional Analyses

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

scholarly journals Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle

1984 ◽  
Vol 223 (3) ◽  
pp. 687-696 ◽  
Author(s):  
C S Harmon ◽  
C G Proud ◽  
V M Pain
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Polypeptide Chain ◽  
Ribosomal Subunit ◽  
Diabetic Rats ◽  
Initiation Factor ◽  
Chain Initiation ◽  
Low Concentrations ◽  
40S Ribosomal Subunit

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.

scholarly journals The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

2012 ◽  
Vol 23 (21) ◽  
pp. 4313-4322 ◽  
Author(s):  
Richa Sardana ◽  
Arlen W. Johnson
Keyword(s):  
Slow Growth ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Small Subunit ◽  
Stable Complex ◽  
Sedimentation Analysis ◽  
Ribosomal Subunits ◽  
Translation Factors ◽  
40S Ribosomal Subunit

We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.

scholarly journals The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae.

2000 ◽  
Vol 47 (4) ◽  
pp. 993-1005 ◽  
Author(s):  
R Gromadka ◽  
J Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Drastic Reduction ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dividing Cells

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.

scholarly journals Deletion of the PAT1 Gene Affects Translation Initiation and Suppresses a PAB1 Gene Deletion in Yeast

2000 ◽  
Vol 20 (10) ◽  
pp. 3538-3549 ◽  
Author(s):  
Françoise Wyers ◽  
Michèle Minet ◽  
Marie Elisabeth Dufour ◽  
Le Thuy Anh Vo ◽  
François Lacroute
Keyword(s):  
Translation Initiation ◽  
Gene Deletion ◽  
Ribosomal Subunit ◽  
Large Decrease ◽  
Free System ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Initiation Of Translation

ABSTRACT The yeast poly(A) binding protein Pab1p mediates the interactions between the 5′ cap structure and the 3′ poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. ThePAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

scholarly journals Citrus exocortis viroid causes ribosomal stress in tomato plants

2019 ◽  
Vol 47 (16) ◽  
pp. 8649-8661 ◽  
Author(s):  
Patrick Cottilli ◽  
Borja Belda-Palazón ◽  
Charith Raj Adkar-Purushothama ◽  
Jean-Pierre Perreault ◽  
Enrico Schleiff ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Tomato Plants ◽  
Translation Machinery ◽  
Citrus Exocortis Viroid ◽  
40S Ribosomal Subunit ◽  
Infected Tomato ◽  
Stress Mediator ◽  
Ribosomal Stress

Abstract Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

scholarly journals Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits

2002 ◽  
Vol 367 (2) ◽  
pp. 359-368 ◽  
Author(s):  
Nilce N. HASHIMOTO ◽  
Larissa S. CARNEVALLI ◽  
Beatriz A. CASTILHO
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
Eukaryotic Initiation Factor ◽  
Strength Of Interaction ◽  
40S Ribosomal Subunit ◽  
Initiator Codon

The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40S ribosomal subunit. In the present study, we have identified amino acid residues in eIF2β required for binding to eIF2γ in yeast. Alteration of six residues in the central region of eIF2β abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of 131Tyr and 132Ser by alanine residues (131YS), although abolishing the binding to eIF2γ in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2γ in vivo was shown by co-immunoprecipitation. The 131YS mutation in eIF2β allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. The combination of this mutation with the 264Ser→Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2β and eIF2γ defines the correct usage of the AUG codon for translation initiation.

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