In Vitro and In Vivo Chemical Labeling of Ribosomal Proteins: A Quantitative Comparison

2012 ◽  
Vol 84 (21) ◽  
pp. 9355-9361 ◽  
Author(s):  
Ethan G. Jaffee ◽  
Matthew A. Lauber ◽  
William E. Running ◽  
James P. Reilly
Keyword(s):  
Ribosomal Proteins ◽  
Quantitative Comparison ◽  
Chemical Labeling

Renal epithelial gene expression profile and bismuth-induced resistance against cisplatin nephrotoxicity

2003 ◽  
Vol 22 (10) ◽  
pp. 535-540 ◽  
Author(s):  
Berend T Leussink ◽  
Hans J Baelde ◽  
Thirza M Broekhuizen-van den Berg ◽  
Emile de Heer ◽  
Gijsbert B van der Voet ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Animal Experiments ◽  
Elongation Factor ◽  
Limiting Factor ◽  
Bismuth Salts ◽  
Proximal Tubular ◽  
A Cell ◽  
Induced Apoptosis

Nephrotoxicity is the most important dose-limiting factor in cisplatin based anti-neoplastic treatment. Pretreatment with bismuth salts, used as pharmaceuticals to treat gastric disorders, has been demonstrated to reduce cisplatin-induced renal cell death in clinical settings and during in vivo and in vitro animal experiments. To investigate the genomic basis of this renoprotective effect, we exposed NRK-52E cells, a cell line of rat proximal tubular epithelial origin, to 33 mM Bi3 for 12 hours, which made them resistant to cisplatin-induced apoptosis. Differentially expressed genes in treated and untreated NRK-52E cells were detected by subtraction PCR and microarray techniques. Genes found to be down regulated (0.17 / 0.31-times) were cytochrome c oxidase subunit I, BAR (an apoptosis regulator), heat-shock protein 70-like protein, and three proteins belonging to the translation machinery (ribosomal proteins S7 and L17, and S1, a member of the elongation factor 1-alpha family). The only up-regulated gene was glutathione Stransferase subunit 3A (1.89-times). Guided by the expression levels of these genes, it may be possible to improve renoprotective treatments during anti-neoplastic therapies.

scholarly journals Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells

1981 ◽  
Vol 194 (3) ◽  
pp. 1007-1009 ◽  
Author(s):  
J Schuck ◽  
G Reichert ◽  
O G Issinger
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Ribosomal Proteins ◽  
Ribosomal Subunits ◽  
Phosphorylated Amino Acid ◽  
Mouse Ascites ◽  
Independent Protein ◽  
Ascites Tumour Cells

Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.

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 Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Cohue Peña ◽  
Sabina Schütz ◽  
Ute Fischer ◽  
Yiming Chang ◽  
Vikram G Panse
Keyword(s):  
Atpase Activity ◽  
Structural Feature ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Spatial Clustering ◽  
Functional Importance ◽  
Eukaryotic Ribosome ◽  
Ribosome Formation

Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome.

scholarly journals Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

1984 ◽  
Vol 4 (8) ◽  
pp. 1605-1617 ◽  
Author(s):  
G Gaines ◽  
G Attardi
Keyword(s):  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
High Efficiency ◽  
Rrna Synthesis ◽  
Isolated Mitochondria ◽  
Highly Efficient ◽  
Light Strand

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

scholarly journals The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Dessie Salilew-Wondim ◽  
Dawit Tesfaye ◽  
Franca Rings ◽  
Eva Held-Hoelker ◽  
Dennis Miskel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Proteins ◽  
Gene Clusters ◽  
Differentially Expressed ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Developmental Capacity ◽  
Eukaryotic Translation Initiation Factors

Abstract Background Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo’s gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones. Results A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3′-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos. Conclusion The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

scholarly journals Quantitative Proteomics Analysis of Berberine-Treated Colon Cancer Cells Reveals Potential Therapy Targets

Biology ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 250
Author(s):  
Pengfei Li ◽  
Zhifang Hao ◽  
Huanhuan Liu ◽  
Bojing Zhu ◽  
Liuyi Dang ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Ribosomal Proteins ◽  
Regulatory Networks ◽  
Mitochondrial Protein ◽  
Tissue Array ◽  
Colon Cancer Cells ◽  
Hub Proteins

Colon cancer is one of the most lethal malignancies worldwide. Berberine has been found to exert potential anti-colon cancer activity in vitro and in vivo, although the detailed regulatory mechanism is still unclear. This study aims to identify the underlying crucial proteins and regulatory networks associated with berberine treatment of colon cancer by using proteomics as well as publicly available transcriptomics and tissue array data. Proteome profiling of berberine-treated colon cancer cells demonstrated that among 5130 identified proteins, the expression of 865 and 675 proteins were changed in berberine-treated HCT116 and DLD1 cells, respectively. Moreover, 54 differently expressed proteins that overlapped in both cell lines were mainly involved in mitochondrial protein synthesis, calcium mobilization, and metabolism of fat-soluble vitamins. Finally, GTPase ERAL1 and mitochondrial ribosomal proteins including MRPL11, 15, 30, 37, 40, and 52 were identified as hub proteins of berberine-treated colon cancer cells. These proteins have higher transcriptional and translational levels in colon tumor samples than that of colon normal samples, and were significantly down-regulated in berberine-treated colon cancer cells. Genetic dependency analysis showed that silencing the gene expression of seven hub proteins could inhibit the proliferation of colon cancer cells. This study sheds a light for elucidating the berberine-related regulatory signaling pathways in colon cancer, and suggests that ERAL1 and several mitochondrial ribosomal proteins might be promising therapeutic targets for colon cancer.

scholarly journals Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different

2021 ◽  
Vol 17 (9) ◽  
pp. e1009949
Author(s):  
Bridget D. De Lay ◽  
Todd A. Cameron ◽  
Nicholas R. De Lay ◽  
Steven J. Norris ◽  
Diane G. Edmondson
Keyword(s):  
Experimental Infection ◽  
Ribosomal Proteins ◽  
Expression Patterns ◽  
Treponema Pallidum ◽  
Transcript Levels ◽  
Transcript Profiles ◽  
High Degree ◽  
Replication Initiator

Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism.

scholarly journals Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

2014 ◽  
Vol 289 (44) ◽  
pp. 30499-30510 ◽  
Author(s):  
Erna Davydova ◽  
Angela Y. Y. Ho ◽  
Jedrzej Malecki ◽  
Anders Moen ◽  
Jorrit M. Enserink ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Sequence Similarity ◽  
Elongation Factor ◽  
Protein Translation ◽  
Cellular Protein ◽  
Yeast Cells ◽  
Lysine Methylation ◽  
Elongation Factor 2

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

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