scholarly journals Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1228 ◽  
Author(s):  
Alexander V. Gopanenko ◽  
Alena V. Kolobova ◽  
Maria I. Meschaninova ◽  
Alya G. Venyaminova ◽  
Alexey E. Tupikin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Protein A ◽  
Hek293 Cells ◽  
Transcription Level ◽  
Noticeable Effect ◽  
Cellular Processes ◽  
The One

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5

1989 ◽  
Vol 9 (12) ◽  
pp. 5281-5288
Author(s):  
W M Wormington
Keyword(s):  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
5S Rrna ◽  
Yeast Cells ◽  
Developmental Expression ◽  
Rrna Synthesis ◽  
Ribosome Assembly ◽  
Subunit Assembly ◽  
Ribosomal Protein L5

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

scholarly journals Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

1989 ◽  
Vol 9 (12) ◽  
pp. 5281-5288 ◽  
Author(s):  
W M Wormington
Keyword(s):  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
5S Rrna ◽  
Yeast Cells ◽  
Developmental Expression ◽  
Rrna Synthesis ◽  
Ribosome Assembly ◽  
Subunit Assembly ◽  
Ribosomal Protein L5

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

Bone Marrow Cells from Patients with Shwachman-Diamond Syndrome Abnormally Express Ribosomal Protein and Ribosomal Biogenesis-Related Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3303-3303
Author(s):  
Piya Rujkijyanont ◽  
Joseph Beyene ◽  
Yigal Dror
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Rna Processing ◽  
Real Time Pcr ◽  
Ribosomal Proteins ◽  
Ribosomal Biogenesis ◽  
Rrna Processing ◽  
Rrna Transcription ◽  
Shwachman Diamond Syndrome ◽  
Marrow Cells

Abstract Background and rational: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by varying degrees of cytopenia and high propensity for myelodysplastic syndrome and acute leukemia. SBDS, the gene associated with SDS, has recently been identified and is postulated to play a role in ribosomal biogenesis and RNA processing, but its functions are still unknown. Defects in ribosomal biogenesis can be characterized by abnormal synthesis of rRNA synthesis or ribosomal proteins or both. Determining the mRNA expression pattern of the various RP genes in SBDS deficient cells will help deciphering the role of SBDS in ribosomal biogenesis. Objectives: To determine whether the primary SDS marrow cells which carry homozygous SBDS mutations abnormally express genes which code for ribosomal proteins (RP) or for proteins that are involved in its transcription. Methods: Total RNA from marrow cells from 9 SDS patients who had hypocellular marrow with normal differential and no malignant transformation and 7 healthy age-matched donors of bone marrows for transplantation was extracted. RNA was labeled and hybridized to Affymetrix HG_U133_Plus2.0 GeneChip. Data were pre-processed using robust multichip analysis (RMA) and differentially expressed genes were identified with permutation-based methods. False discovery rate (FDR)-adjusted p-values were used to rank genes and cluster analysis grouped genes and samples. T-statistic values were used to screen for differentially expressed RP-related genes. Real-time PCR was performed to confirm differential expression of genes found by oligonucleotide microarray. Results: Of the 38,500 genes on the HG_133_Plus2.0 we analyzed 375 known ribosomal protein and RNA processing-related genes. Interestingly, there were differences in the expression pattern of the RP genes, suggesting differential regulation of these genes in Sbds-deficient cells. Interestingly, despite uniform decrease in RP gene expression in reduced cell growth conditions, only 27 of the 85 RP genes were downregulated. Downregulation of representative 2 genes was confirmed by real-time PCR. Further, one of the RP genes, RPL27L was upregulated. This gene, which is a target of p53, has a non-ribosomal function and lead to accelerated apoptosis. It is noteworthy that several genes involved in mRNA transcription such as GABPA and YY1were downregulated without dysregulation of genes involved in mRNA degradation, suggesting that the downregulation of the RP gene expression is at the transcription level. In addition to dysregulation of the RP mRNA we also found dysregulation of genes involved in rRNA transcription (e.g. MKI67IP) and pre rRNA processing (e.g. FBL). Conclusions: SBDS-deficiency results in dysregulation of selective group of RP genes as well as genes related to rRNA processing and rRNA transcription. Future studies should focus on the mechanism of the abnormal expression as well as its biological consequences.

scholarly journals Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

2020 ◽  
Author(s):  
Matthew Stuible ◽  
Christian Gervais ◽  
Simon Lord-Dufour ◽  
Sylvie Perret ◽  
Denis L’Abbe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Transient Gene Expression ◽  
Full Length ◽  
Spike Protein ◽  
Hek293 Cells ◽  
High Yield ◽  
Diagnostic Tools ◽  
Purification Method

ABSTRACTRecombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

scholarly journals Autoregulation of yeast ribosomal proteins discovered by efficient search for feedback regulation

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Basab Roy ◽  
David Granas ◽  
Fredrick Bragg ◽  
Jonathan A. Y. Cher ◽  
Michael A. White ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Ribosomal Proteins ◽  
Feedback Regulation ◽  
Ribosomal Protein Genes ◽  
Efficient Search ◽  
Fold Reduction

AbstractPost-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes. We used the yeast collection of genes fused to GFP as a rapid screen for examples of feedback regulation in ribosomal proteins by overexpressing a non-regulatable version of a gene and observing the effects on the expression of the GFP-fused version. We tested 95 ribosomal protein genes and found a wide continuum of effects, with 30% showing at least a 3-fold reduction in expression. Two genes, RPS22B and RPL1B, showed over a 10-fold repression. In both cases the cis-regulatory segment resides in the 5’ UTR of the gene as shown by placing that segment of the mRNA upstream of GFP alone and demonstrating it is sufficient to cause repression of GFP when the protein is over-expressed. Further analyses showed that the intron in the 5’ UTR of RPS22B is required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5’ UTR of RPL1B contains a sequence and structure motif that is conserved in the binding sites of Rpl1 orthologs from bacteria to mammals, and mutations within the motif eliminate repression.

scholarly journals Identification of Phosphotyrosine Binding Domain-Containing Proteins as Novel Downstream Targets of the EphA8 Signaling Function

2007 ◽  
Vol 27 (23) ◽  
pp. 8113-8126 ◽  
Author(s):  
Jongdae Shin ◽  
Changkyu Gu ◽  
Eunjeong Park ◽  
Soochul Park
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Dominant Negative Mutant ◽  
Eph Receptors ◽  
Protein Protein Interaction ◽  
Cellular Processes ◽  
Phosphotyrosine Binding Domain ◽  
Ptb Domain

ABSTRACT Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway.

scholarly journals Ribosomal proteins: mutant phenotypes by the numbers and associated gene expression changes

Open Biology ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 200114
Author(s):  
Michael Polymenis
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Homo Sapiens ◽  
Ribosome Profiling ◽  
Ribosomal Protein Genes ◽  
Future Studies ◽  
Protein Mutants ◽  
Structural Parts ◽  
Mutant Phenotypes

Ribosomal proteins are highly conserved, many universally so among organisms. All ribosomal proteins are structural parts of the same molecular machine, the ribosome. However, when ribosomal proteins are mutated individually, they often lead to distinct and intriguing phenotypes, including specific human pathologies. This review is an attempt to collect and analyse all the reported phenotypes of each ribosomal protein mutant in several eukaryotes ( Saccharomyces cerevisiae , Caenorhabditis elegans , Drosophila melanogaster , Danio rerio , Mus musculus , Homo sapiens ). These phenotypes were processed with unbiased computational approaches to reveal associations between different phenotypes and the contributions of individual ribosomal protein genes. An overview of gene expression changes in ribosomal protein mutants, with emphasis on ribosome profiling studies, is also presented. The available data point to patterns that may account for most of the observed phenotypes. The information presented here may also inform future studies about the molecular basis of the phenotypes that arise from mutations in ribosomal proteins.

scholarly journals Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

1989 ◽  
Vol 9 (9) ◽  
pp. 3679-3684 ◽  
Author(s):  
S Huang ◽  
J W Hershey
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Protein Gene ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Translational Initiation ◽  
Ribosomal Protein Synthesis ◽  
Translational Initiation Factor

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.

scholarly journals New Tricks for an old molecule: Preserved antibacterial activity of ribosomal protein S15 during evolution

2020 ◽  
Author(s):  
Baozhen Qu ◽  
Zengyu Ma ◽  
Lan Yao ◽  
Zhan Gao ◽  
Shicui Zhang
Keyword(s):  
Antibacterial Activity ◽  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Critical Role ◽  
Circulation System ◽  
Bacterial Membrane ◽  
Peptide Antibiotics ◽  
Gram Negative ◽  
First Time

AbstractPrevious studies show that some ribosomal proteins possess antimicrobial peptide (AMP) activity. However, information as such remains rather fragmentary and limited. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial membrane via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel peptide antibiotics against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.Author summaryRibosomal protein, a component of ribonucleoprotein particles, is traditionally known involved in protein synthesis in a cell. Here we demonstrated for the first time that amphioxus ribosomal protein 15 was a novel antibacterial protein, capable of recognizing Gram-negative and -positive bacteria as well as killing them. It killed the bacteria by a combined mode of action of disrupting bacterial membrane integrity and inducing radical oxygen species production. We also showed that both eukaryotic ribosomal protein 15 and its prokaryotic counterpart ribosomal protein 19 possessed antibacterial activity, indicating that the antibacterial property is universal for this family of molecules. Moreover, we found that ribosomal protein 15 was present in the circulation system of various animals including shrimp, amphioxus, zebrafish and mice, suggesting it may physiologically play a key role in systematic immunity. Altogether, our study provides a new angle for understanding the biological function of ribosomal proteins.

Expression and affinity purification of recombinant mammalian mitochondrial ribosomal small subunit (MRPS) proteins and protein–protein interaction analysis indicate putative role in tumourigenic cellular processes

2021 ◽  
Author(s):  
Oviya Revathi Paramasivam ◽  
Gopal Gopisetty ◽  
Jayavelu Subramani ◽  
Rajkumar Thangarajan
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Ribosomal Proteins ◽  
Interaction Analysis ◽  
Affinity Purification ◽  
Small Subunit ◽  
Acid Oxidation ◽  
Expression Screening ◽  
Cellular Processes ◽  
Putative Role

Abstract Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein–protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.

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