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The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs.

ABSTRACT: Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational 'closed loop' complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions.

SUBMITTER: Thompson MK

PROVIDER: S-EPMC4848094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs

Project description:Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions.

2016-04-24 | GSE61753 | GEO

Ribosomal protein RACK1 enhances translation of poliovirus and other viral IRESs.

Project description:Viruses have evolved strategies to ensure efficient translation using host cell ribosomes and translation factors. In addition to cleaving translation initiation factors required for host cell translation, poliovirus (PV) uses an internal ribosome entry site (IRES). Recent studies suggest that viruses exploit specific ribosomal proteins to enhance translation of their viral proteins. The ribosomal protein receptor for activated C kinase 1 (RACK1), a protein of the 40S ribosomal subunit, was previously shown to mediate translation from the 5' cricket paralysis virus and hepatitis C virus IRESs. Here we found that translation of a PV dual-luciferase reporter shows a moderate dependence on RACK1. However, in the context of a viral infection we observed significantly reduced poliovirus plaque size and titers and delayed host cell translational shut-off. Our findings further illustrate the involvement of the cellular translational machinery during PV infection and how viruses usurp the function of specific ribosomal proteins.

| S-EPMC7804209 | biostudies-literature

Communication between RACK1/Asc1 and uS3 (Rps3) is essential for RACK1/Asc1 function in yeast Saccharomyces cerevisiae.

Project description:The receptor for activated c-kinase (RACK1, Asc1 in yeast) is a eukaryotic ribosomal protein located in the head region of the 40S subunit near the mRNA exit channel. This WD-repeat ?-propeller protein acts as a signaling molecule and is involved in metabolic regulation, cell cycle progression, and translational control. However, the exact details of the RACK1 recruitment and stable association with the 40S ribosomal subunit remain only partially known. X-ray analyses of the yeast, Saccharomyces cerevisiae, ribosome revealed that the RACK1 propeller blade (4-5) interacts with the eukaryote-specific C-terminal domain (CTD) of ribosomal protein S3 (uS3 family). To check the functional significance of this interaction, we generated mutant yeast strains harboring C-terminal deletions of uS3. We found that deletion of the 20 C-terminal residues (interacting with blade 4-5) from the uS3-CTD abrogates RACK1 binding to the ribosome. Strains with truncated uS3-CTD exhibited compromised cellular growth and protein synthesis similar to that of RACK1? strain, thus suggesting that the uS3-CTD is crucial not only for the recruitment and association of RACK1 with the ribosome, but also for its intracellular function. We suggest that eukaryote-specific RACK1-uS3 interaction has evolved to act as a link between the ribosome and the cellular signaling pathways.

| S-EPMC6538471 | biostudies-literature

The eIF3c/NIP1 PCI domain interacts with RNA and RACK1/ASC1 and promotes assembly of translation preinitiation complexes.

Project description:Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1-3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein-protein binding domain in mediating protein-RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.

| S-EPMC3315329 | biostudies-literature

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1.

Project description:Heterotrimeric G proteins were originally discovered through efforts to understand the effects of hormones, such as glucagon and epinephrine, on glucose metabolism. On the other hand, many cellular metabolites, including glucose, serve as ligands for G protein-coupled receptors. Here we investigate the consequences of glucose-mediated receptor signaling, and in particular the role of a Gα subunit Gpa2 and a non-canonical Gβ subunit, known as Asc1 in yeast and RACK1 in animals. Asc1/RACK1 is of particular interest because it has multiple, seemingly unrelated, functions in the cell. The existence of such "moonlighting" operations has complicated the determination of phenotype from genotype. Through a comparative analysis of individual gene deletion mutants, and by integrating transcriptomics and metabolomics measurements, we have determined the relative contributions of the Gα and Gβ protein subunits to glucose-initiated processes in yeast. We determined that Gpa2 is primarily involved in regulating carbohydrate metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Of the two subunits, Gpa2 regulates a greater number of gene transcripts and was particularly important in determining the amplitude of response to glucose addition. We conclude that the two G protein subunits regulate distinct but complementary processes downstream of the glucose-sensing receptor, as well as processes that lead ultimately to changes in cell growth and metabolism.

| S-EPMC8282090 | biostudies-literature

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Project description:Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

| S-EPMC3128278 | biostudies-literature

Ribosome-associated Asc1/RACK1 is required for endonucleolytic cleavage induced by stalled ribosome at the 3' end of nonstop mRNA.

Project description:Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3' ends, and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3' end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3' end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells, and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells, and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA.

| S-EPMC4911565 | biostudies-literature

Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.

Project description:Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.

| S-EPMC3858243 | biostudies-literature

Alteration of a novel dispensable mitochondrial ribosomal small-subunit protein, Rsm28p, allows translation of defective COX2 mRNAs.

Project description:Mutations affecting the RNA sequence of the first 10 codons of the Saccharomyces cerevisiae mitochondrial gene COX2 strongly reduce translation of the mRNA, which encodes the precursor of cytochrome c oxidase subunit II. A dominant chromosomal mutation that suppresses these defects is an internal in-frame deletion of 67 codons from the gene YDR494w. Wild-type YDR494w encodes a 361-residue polypeptide with no similarity to proteins of known function. The epitope-tagged product of this gene, now named RSM28, is both peripherally associated with the inner surface of the inner mitochondrial membrane and soluble in the matrix. Epitope-tagged Rsm28p from Triton X-100-solubilized mitochondria sedimented with the small subunit of mitochondrial ribosomes in a sucrose gradient containing 500 mM NH4Cl. Complete deletion of RSM28 caused only a modest decrease in growth on nonfermentable carbon sources in otherwise wild-type strains and enhanced the respiratory defect of the suppressible cox2 mutations. The rsm28 null mutation also reduced translation of an ARG8m reporter sequence inserted at the COX1, COX2, and COX3 mitochondrial loci. We tested the ability of RSM28-1 to suppress a variety of cox2 and cox3 mutations and found that initiation codon mutations in both genes were suppressed. We conclude that Rsm28p is a dispensable small-subunit mitochondrial ribosomal protein previously undetected in systematic investigations of these ribosomes, with a positive role in translation of several mitochondrial mRNAs.

| S-EPMC549345 | biostudies-literature

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs.

Project description:In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.

| S-EPMC305564 | biostudies-literature

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