scholarly journals Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mehdi Eshraghi ◽  
Pabalu P. Karunadharma ◽  
Juliana Blin ◽  
Neelam Shahani ◽  
Emiliano P. Ricci ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Huntington Disease ◽  
Ribosomal Proteins ◽  
Mutant Huntingtin ◽  
Translation Elongation ◽  
Polyglutamine Expansion ◽  
Ribosome Stalling ◽  
Mrna Targets ◽  
Discernible Effect

AbstractThe polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis in mouse HD striatal neuronal cells. Depletion of mHtt enhances protein synthesis and increases the speed of ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro. Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but its depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicates a widespread shift in ribosome occupancy toward the 5′ and 3′ end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation elongation, a mechanistic defect that can be exploited for HD therapeutics.

scholarly journals Interaction of Huntington Disease Protein with Transcriptional Activator Sp1

2002 ◽  
Vol 22 (5) ◽  
pp. 1277-1287 ◽  
Author(s):  
Shi-Hua Li ◽  
Anna L. Cheng ◽  
Hui Zhou ◽  
Suzanne Lam ◽  
Manjula Rao ◽  
...  
Keyword(s):  
Huntington Disease ◽  
Cultured Cells ◽  
Growth Factor Receptor ◽  
Soluble Form ◽  
Mutant Huntingtin ◽  
Nuclear Effect ◽  
Polyglutamine Expansion ◽  
Disease Protein ◽  
Cellular Dysfunction

ABSTRACT Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. Here we report that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes.

scholarly journals Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Mikhail V. Nesterchuk ◽  
Elena M. Smekalova ◽  
Joao A. Paulo ◽  
Piotr S. Kowalski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Mouse Liver ◽  
Negative Impact ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Elongation Factor 2

Abstract Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

scholarly journals Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein

2019 ◽  
pp. 201812056
Author(s):  
Ying Yang ◽  
Yang Geng ◽  
Dongyun Jiang ◽  
Lin Ning ◽  
Hyung Joon Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Alternative Pathways ◽  
Fragile X Mental Retardation ◽  
Mrna Targets ◽  
Mental Retardation Protein ◽  
Pathway Inhibition

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP–mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

scholarly journals THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
Author(s):  
Paul M. Lizardi ◽  
David J. L. Luck
Keyword(s):  
Protein Synthesis ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Selective Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

scholarly journals P11.36 Ribosome hydroxylase Mina53 is required for Glioblastoma and is involved in regulation of translation rateand fidelity by regulating ribosomal biogenesis

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii51-iii51
Author(s):  
D Pandey ◽  
F Mohammad ◽  
S Weissmann ◽  
P Hallenborg ◽  
B Blagoev ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
High Throughput Sequencing ◽  
Affinity Purification ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Ribosomal Biogenesis ◽  
Isolation And Identification ◽  
Knock Down

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive types of tumors with a poor response to standard treatment and a median 5-year survival of less than 5%. Therefore, there is an urgent need for new treatments. Recently, a large number of genome-wide studies have shown that the epigenetic modifiers are frequently deregulated in cancer. Using a mouse GBM model, we performed in vitro and in vivo shRNA screens to identify epigenetic regulators required for the tumorigenic process in GBM. Among these regulators is a ribosome hydroxylase Mina53 which hydroxylates His-39 of ribosomal protein, RPL27a. We have found that the knock-down (KD) of Mina53 reduces the in vitro proliferation and colony forming ability of mouse glioma initiating cells (mGIC) and this is dependent on the catalytic activity of Mina. Knock-down of Mina resulted into a small but significant reduction in the global protein synthesis rate. A tandem affinity purification experiment to identify proteins associated with Mina revealed that it is associated mainly with ribosomal proteins, including its substrate RPL27a. Global proteomic analyses revealed that final amounts and de novo protein synthesis of many ribosomal proteins were reduced upon Mina depletion. Isolation and identification of different polysome fraction bound mRNAs using high-throughput sequencing found that mRNAs encoding many ribosomal proteins have lower number of ribosomes loaded on them in the Mina depleted samples compared to the control. Taken together, this study has found that Mina53 is required for glioblastoma and it regulates translation through regulation of ribosomal biogenesis

Suppressors suaC 109 and suaA 101 of Aspergillus nidulans alter the ribosomal phenotype in vitro

1987 ◽  
Vol 7 (12) ◽  
pp. 941-948 ◽  
Author(s):  
A. Zamir ◽  
S. S. Martinelli
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Ribosomal Proteins ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Cell Free System ◽  
Translation Factors ◽  
Acid Incorporation

A new homologous, cell-free system for protein synthesis has been devised for use with ribosomes and elongation factors from Aspergillus nidulans. Ribosome preparations from strains with either the suaAlO1 or suaCl09 mutations have a higher misreading ratio (non-cognate:cognate amino acid incorporation) in the presence of hygromycin than controls. They can be classed as fidelity mutants. These results also prove that the mutations must be in genes coding for ribosomal proteins or enzymes which modify ribosomal proteins post-translationally. Alternatively, the genes could code for translation factors.

scholarly journals In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

2021 ◽  
Vol 4 (3) ◽  
pp. 45
Author(s):  
Riku Nagai ◽  
Yichen Xu ◽  
Chang Liu ◽  
Ayaka Shimabukuro ◽  
Nono Takeuchi-Tomita
Keyword(s):  
Unnatural Amino Acids ◽  
Molecular Mechanisms ◽  
In Vitro Translation ◽  
Critical Parameters ◽  
Translation System ◽  
Reaction Conditions ◽  
Translation Elongation ◽  
Functional Studies ◽  
Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

scholarly journals Overexpression of Translation Elongation Factor 1A Affects the Organization and Function of the Actin Cytoskeleton in Yeast

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1425-1436
Author(s):  
Raj Munshi ◽  
Kimberly A Kandl ◽  
Anne Carr-Schmid ◽  
Johanna L Whitacre ◽  
Alison E M Adams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

Abstract The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1Bα, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

scholarly journals What determines eukaryotic translation elongation: recent molecular and quantitative analyses of protein synthesis

Open Biology ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 200292
Author(s):  
Nagammal Neelagandan ◽  
Irene Lamberti ◽  
Hugo J. F. Carvalho ◽  
Cédric Gobet ◽  
Felix Naef
Keyword(s):  
Protein Synthesis ◽  
Single Cells ◽  
Comprehensive Overview ◽  
Translation Elongation ◽  
Dynamic Regulation ◽  
Eukaryotic Translation ◽  
Ribosome Stalling ◽  
Theoretical Approaches ◽  
Genome Wide ◽  
Quantitative Analyses

Protein synthesis from mRNA is an energy-intensive and tightly controlled cellular process. Translation elongation is a well-coordinated, multifactorial step in translation that undergoes dynamic regulation owing to cellular state and environmental determinants. Recent studies involving genome-wide approaches have uncovered some crucial aspects of translation elongation including the mRNA itself and the nascent polypeptide chain. Additionally, these studies have fuelled quantitative and mathematical modelling of translation elongation. In this review, we provide a comprehensive overview of the key determinants of translation elongation. We discuss consequences of ribosome stalling or collision, and how the cells regulate translation in case of such events. Next, we review theoretical approaches and widely used mathematical models that have become an essential ingredient to interpret complex molecular datasets and study translation dynamics quantitatively. Finally, we review recent advances in live-cell reporter and related analysis techniques, to monitor the translation dynamics of single cells and single-mRNA molecules in real time.

scholarly journals 307CHARACTERIZATION OF PROTEIN PHOSPHORYLATIONS IN THE COURSE OF MEIOTIC MATURATION OF BOVINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 273
Author(s):  
M. Bhojwani ◽  
M. Marx ◽  
F. Melo-Sterza ◽  
W. Kanitz ◽  
C. Leiding ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Phosphorylated Proteins ◽  
Stage Of Development ◽  
Peptide Mass ◽  
Fluorescence Dye ◽  
Bovine Oocytes

The importance of protein phosphorylations during meiotic maturation (transition from prophase I to metaphase II) of oocytes is documented by the fact that the inhibition of the M-phase kinases, cdc2k or MAPK, arrests the oocytes in the GV stage. A detailed knowledge of the targets of these kinases during this stage of development is still missing. Therefore, we have analyzed the proteome of bovine oocytes by high resolution 2D-gel electrophoresis to detect differences in the expression and phosphorylation state of proteins in the course of in vitro maturation (IVM). Bovine oocytes were matured for different times in TCM 199 containing 3% BSA and 300 oocytes each in GV stage (0-h maturation), in GVBD/M I (10-h maturation) or in M II stage (240h maturation) were separated on the gels. The proteins were visualized by staining them with silver or with the fluorescence dye Sypro Ruby, and phosphorylated proteins were detected by Western Blotting with Ser-, Thr-, or Tyr-phosphorylation specific antibodies or by staining with the phosphoprotein specific fluorescence dye Pro-Q Diamond. Gels made from oocytes at the above mentioned maturation stages were compared by a computerized gel-overlay software program (2D Decodon, Greitswald, Germany). The overall protein synthesis was statistically analysed by ANOVA (SigmaStat, Ekrath, Germany), pairwise multiple comparison procedure. Only distinct spots with a difference greater than 30% in their optical densities were considered to be differently expressed or phosphorylated. The results showed a three-fold increase in the rate of overall protein synthesis (p 0.05) during GVBD. Newly synthesized proteins were detected mainly in the higher molecular weight (MW) range (60–80kDa), and protein degradations were found mainly in the lower MW range (20–40kDa) after GVBD. Preliminary data obtained by analyzing the phosphorylation pattern showed that obviously no phosphorylated proteins could be detected in the GV-stage oocytes. Phosphorylation of different proteins was observed at the time of GVBD after 6 to 10h IVM, concomitantly with the activation of cdc2k and MAPK. A maximum of phosphorylated proteins was observed in metaphase II. The first results obtained by performing peptide mass fingerprinting using MALDI-Tof showed that members of the family of heat-shock proteins, ribosomal proteins and putative zinc finger proteins (transcription regulators) were differently expressed or phosphorylated during IVM. This work was supported by the DFG, To 178/1-1, 2 and by the Eibl-Stiftung.

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