scholarly journals Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins

2020 ◽  
pp. 57-57
Author(s):  
Bogdan Jovanovic ◽  
Lisa Schubert ◽  
Fabian Poetz ◽  
Georg Stoecklin
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Associated Factors ◽  
Ribosomal Subunit ◽  
High Specificity ◽  
Negative Control ◽  
Tev Protease ◽  
Protease Cleavage ◽  
Associated Proteins ◽  
And Function

Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.

scholarly journals A DEAD-box protein regulates ribosome assembly through control of ribosomal protein synthesis

2019 ◽  
Vol 47 (15) ◽  
pp. 8193-8206 ◽  
Author(s):  
Isabelle Iost ◽  
Chaitanya Jain
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Large Family ◽  
Ribosome Assembly ◽  
Protein Abundance ◽  
Ribosomal Assembly ◽  
Dead Box ◽  
Ribosomal Protein Synthesis ◽  
Intrinsic Transcription Termination

Abstract DEAD-box proteins (DBPs) comprise a large family of proteins that most commonly have been identified as regulators of ribosome assembly. The Escherichia coli DBP, SrmB, represents a model bacterial DBP whose absence impairs formation of the large ribosomal subunit (LSU). To define the basis for SrmB function, suppressors of the ribosomal defect of ΔsrmB strains were isolated. The major class of suppressors was found to map to the 5′ untranslated region (UTR) of the rplM-rpsI operon, which encodes the ribosomal proteins (r-proteins) L13 and S9. An analysis of protein abundance indicated that both r-proteins are under-produced in the ΔsrmB strain, but are increased in these suppressors, implicating r-protein underproduction as the molecular basis for the observed ribosomal defects. Reduced r-protein synthesis was determined to be caused by intrinsic transcription termination within the rplM 5′ UTR that is abrogated by SrmB. These results reveal a specific mechanism for DBP regulation of ribosomal assembly, indirectly mediated through its effects on r-protein expression.

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

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1035 ◽  
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.

scholarly journals Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques

2007 ◽  
Vol 189 (9) ◽  
pp. 3434-3444 ◽  
Author(s):  
M. Jiang ◽  
S. M. Sullivan ◽  
A. K. Walker ◽  
J. R. Strahler ◽  
P. C. Andrews ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Protein Identification ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Absolute Quantitation ◽  
Proteomic Approach ◽  
Assembly Factors ◽  
Associated Proteins ◽  
Isobaric Tags

ABSTRACT Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16°C and 37°C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

scholarly journals The Antituberculosis Antibiotic Capreomycin Inhibits Protein Synthesis by Disrupting Interaction between Ribosomal Proteins L12 and L10

2014 ◽  
Vol 58 (4) ◽  
pp. 2038-2044 ◽  
Author(s):  
Yuan Lin ◽  
Yan Li ◽  
Ningyu Zhu ◽  
Yanxing Han ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Multiple Drug ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Multiple Drug Resistant ◽  
Ribosomal Function ◽  
Line Drug

ABSTRACTCapreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin.Mycobacterium tuberculosisribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 inM. smegmatis, a species close toM. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

scholarly journals Altered ribosomal function and protein synthesis caused by tau

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Harrison Tudor Evans ◽  
Deonne Taylor ◽  
Andrew Kneynsberg ◽  
Liviu-Gabriel Bodea ◽  
Jürgen Götz
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
De Novo ◽  
Ribosomal Subunit ◽  
Fundamental Aspect ◽  
Amino Terminal ◽  
Polysome Profiling ◽  
Ribosomal Function ◽  
Neurological Processes ◽  
Projection Domain

AbstractThe synthesis of new proteins is a fundamental aspect of cellular life and is required for many neurological processes, including the formation, updating and extinction of long-term memories. Protein synthesis is impaired in neurodegenerative diseases including tauopathies, in which pathology is caused by aberrant changes to the microtubule-associated protein tau. We recently showed that both global de novo protein synthesis and the synthesis of select ribosomal proteins (RPs) are decreased in mouse models of frontotemporal dementia (FTD) which express mutant forms of tau. However, a comprehensive analysis of the effect of FTD-mutant tau on ribosomes is lacking. Here we used polysome profiling, de novo protein labelling and mass spectrometry-based proteomics to examine how ribosomes are altered in models of FTD. We identified 10 RPs which were decreased in abundance in primary neurons taken from the K3 mouse model of FTD. We further demonstrate that expression of human tau (hTau) decreases both protein synthesis and biogenesis of the 60S ribosomal subunit, with these effects being exacerbated in the presence of FTD-associated tau mutations. Lastly, we demonstrate that expression of the amino-terminal projection domain of hTau is sufficient to reduce protein synthesis and ribosomal biogenesis. Together, these data reinforce a role for tau in impairing ribosomal function.

p70 Ribosomal protein S6 kinase (Rps6kb1): an update

2014 ◽  
Vol 67 (12) ◽  
pp. 1019-1025 ◽  
Author(s):  
Farnaz Bahrami-B ◽  
Parvin Ataie-Kachoie ◽  
Mohammad H Pourgholami ◽  
David L Morris
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Mtor Inhibitors ◽  
Therapeutic Index ◽  
Drug Induced ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Ribosomal Protein S6 Kinase

The Rps6kb1 gene encodes the 70 kDa ribosomal protein S6 kinase (p70S6K), which is a serine/threonine kinase regulated by phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. p70S6K plays a crucial role in controlling cell cycle, growth and survival. The PI3K/mTOR signalling pathway is one of the major mechanisms for controlling cell survival, proliferation and metabolism and is the central regulator of translation of some components of protein synthesis system. Upon activation, this kinase phosphorylates S6 protein of ribosomal subunit 40S resulting in selective translation of unique family of mRNAs that contain oligopyrimidine tract on 5’ transcriptional site (5′TOP). 5′TOP mRNAs are coding the components of translational apparatus including ribosomal proteins and elongation factors. Due to the role of p70S6K in protein synthesis and also its involvement in a variety of human diseases ranging from diabetes and obesity to cancer, p70S6K is now being considered as a new therapeutic target for drug development. Furthermore, p70S6K acts as a biomarker for response to immunosuppressant as well as anticancer effects of inhibitors of the mTOR. Because of the narrow therapeutic index of mTOR inhibitors, drug monitoring is essential, and this is usually done by measuring blood drug levels, therapeutic response and drug-induced adverse effects. Recent studies have suggested that plasma p70S6K is a reliable index for the monitoring of patient response to mTOR inhibitors. Therefore, a better understanding of p70S6K and its role in various pathological conditions could enable the development of strategies to aid diagnosis, prognosis and treatment schedules.

Structure of rRNA in the ribosome

1992 ◽  
Vol 50 (1) ◽  
pp. 462-463
Author(s):  
M. Boublik ◽  
J. S. Wall
Keyword(s):  
Ribosomal Proteins ◽  
Molecular Level ◽  
Protein Biosynthesis ◽  
Ribosomal Subunit ◽  
Structure And Function ◽  
Ribonucleic Acids ◽  
Subcellular Organelles ◽  
Fundamental Relationship ◽  
And Function ◽  
Internal Architecture

Ribosomes are complex subcellular organelles playing a central role in protein biosynthesis. They are composed of more than fifty different proteins and three or four ribonucleic acids (rRNA) unevenly distributed (with no symmetry) between the large and small ribosomal subunit. It has been well established that ribosomal proteins and rRNAs are both involved in formation of the internal architecture of the ribosome as well as its function in protein synthesis. Understanding the fundamental relationship between structure and function requires establishment of the 3-D structure of the ribosome and its components at a molecular level.

scholarly journals From differential transcription of ribosomal proteins to differential structure of ribosomes

2015 ◽  
Author(s):  
Nikolai Slavov
Keyword(s):  
Growth Rate ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Ribosomal Proteins ◽  
Structure And Function ◽  
Mrna Levels ◽  
Cell Growth Rate ◽  
Direct Data ◽  
And Function ◽  
Differential Transcription

About a decade ago, I observed that as the cell growth rate increases, mRNAs coding for ribosomal proteins are transcriptionally induced to varying degrees. This observation puzzled me as it defied my expectation that faster growing cells meet their demands for increased protein synthesis by simply inducing all ribosomal proteins to the same degree to make more ribosomes. These initial data were limited to mRNA levels and thus too indirect to make concrete conclusions about ribosomal structure and function. This commentary outlines my trajectory investigating this puzzle in search of more direct data.

scholarly journals From differential transcription of ribosomal proteins to differential structure of ribosomes

2015 ◽  
Author(s):  
Nikolai Slavov
Keyword(s):  
Growth Rate ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Ribosomal Proteins ◽  
Structure And Function ◽  
Mrna Levels ◽  
Cell Growth Rate ◽  
Direct Data ◽  
And Function ◽  
Differential Transcription

About a decade ago, I observed that as the cell growth rate increases, mRNAs coding for ribosomal proteins are transcriptionally induced to varying degrees. This observation puzzled me as it defied my expectation that faster growing cells meet their demands for increased protein synthesis by simply inducing all ribosomal proteins to the same degree to make more ribosomes. These initial data were limited to mRNA levels and thus too indirect to make concrete conclusions about ribosomal structure and function. This commentary outlines my trajectory investigating this puzzle in search of more direct data.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Sign in / Sign up

Export Citation Format

Share Document