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Journal articles on the topic 'Protein and mRNA levels'
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1
Berger, Lloyd C., Jnanankur Bag, and Bruce H. Sells. "Translation of poly(A)-binding protein mRNA is regulated by growth conditions." Biochemistry and Cell Biology 70, no. 9 (September 1, 1992): 770–78. http://dx.doi.org/10.1139/o92-117.
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Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (~ 60%) and resting cells (~ 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5′ untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.Key words: translation, mRNP, poly(A) binding protein.
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Gerstenfeld, L. C., M. H. Finer, and H. Boedtker. "Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts." Molecular and Cellular Biology 5, no. 6 (June 1985): 1425–33. http://dx.doi.org/10.1128/mcb.5.6.1425-1433.1985.
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Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.
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Gerstenfeld, L. C., M. H. Finer, and H. Boedtker. "Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts." Molecular and Cellular Biology 5, no. 6 (June 1985): 1425–33. http://dx.doi.org/10.1128/mcb.5.6.1425.
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Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.
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Willis, Dianna E., Erna A. van Niekerk, Yukio Sasaki, Mariano Mesngon, Tanuja T. Merianda, Gervan G. Williams, Marvin Kendall, Deanna S. Smith, Gary J. Bassell, and Jeffery L. Twiss. "Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs." Journal of Cell Biology 178, no. 6 (September 4, 2007): 965–80. http://dx.doi.org/10.1083/jcb.200703209.
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Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.
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Veletza, S. V., K. V. Nichols, I. Gross, H. Lu, D. W. Dynia, and J. Floros. "Surfactant protein C: hormonal control of SP-C mRNA levels in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 6 (June 1, 1992): L684—L687. http://dx.doi.org/10.1152/ajplung.1992.262.6.l684.
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We have studied hormonal regulation of the surfactant protein C (SP-C) in fetal 18-dah rat lung explants. SP-C mRNA was detected in Northern blots with a specific rat SP-C cDNA probe and quantified by densitometry. Treatment of the explants with dexamethasone resulted in a dose-dependent increase of the SP-C mRNA level. Transcriptional assays have shown that the regulation of SP-C mRNA by dexamethasone involves a transcriptional step. Administration of the cAMP analogues, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), produced a dose-dependent increase of SP-C mRNA levels, with maximum stimulation observed at 200 microM. The thyroid hormone T3 had no effect on SP-C mRNA levels, whether administered alone or in combination with dexamethasone. Variation in the effects of the above hormones on three surfactant protein mRNAs, SP-A, SP-B and SP-C, indicates that the hormonal regulation of the surfactant proteins is a complex process and that each gene is, in part, differentially regulated.
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Maicas, E., F. G. Pluthero, and J. D. Friesen. "The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay." Molecular and Cellular Biology 8, no. 1 (January 1988): 169–75. http://dx.doi.org/10.1128/mcb.8.1.169-175.1988.
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The suggestion that compensation for overabundant mRNA of the genes for Saccharomyces cerevisiae ribosomal protein (r-protein) L3, L29, or rp59 occurs by translation repression has been reinvestigated. First, analysis of the distribution of these three mRNAs in polysome profiles revealed no differences between normal and mRNA-overproducing strains, indicating that initiation of r-protein translation is not repressed under conditions of mRNA overaccumulation. Second, experiments involving radioactive pulse-labeling of proteins were done by using a modified method of data collection and analysis that allows quantitation and correction for fast decay during the pulse. These measurements revealed that the synthesis rate of the three r-proteins is increased when their mRNA levels are elevated and that their decay rate is also high, with half-lives ranging from a fraction of a minute to more than 10 min. We conclude that accumulation of excess r-protein mRNA has no effect on translation rate; rapid decay of protein during the course of the labeling period can account for the apparent discrepancy between mRNA levels and protein synthesis rates. Yeast r-proteins, when produced in excess, are among the most rapidly degraded proteins so far described.
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Maicas, E., F. G. Pluthero, and J. D. Friesen. "The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay." Molecular and Cellular Biology 8, no. 1 (January 1988): 169–75. http://dx.doi.org/10.1128/mcb.8.1.169.
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The suggestion that compensation for overabundant mRNA of the genes for Saccharomyces cerevisiae ribosomal protein (r-protein) L3, L29, or rp59 occurs by translation repression has been reinvestigated. First, analysis of the distribution of these three mRNAs in polysome profiles revealed no differences between normal and mRNA-overproducing strains, indicating that initiation of r-protein translation is not repressed under conditions of mRNA overaccumulation. Second, experiments involving radioactive pulse-labeling of proteins were done by using a modified method of data collection and analysis that allows quantitation and correction for fast decay during the pulse. These measurements revealed that the synthesis rate of the three r-proteins is increased when their mRNA levels are elevated and that their decay rate is also high, with half-lives ranging from a fraction of a minute to more than 10 min. We conclude that accumulation of excess r-protein mRNA has no effect on translation rate; rapid decay of protein during the course of the labeling period can account for the apparent discrepancy between mRNA levels and protein synthesis rates. Yeast r-proteins, when produced in excess, are among the most rapidly degraded proteins so far described.
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Chen, Xu-Qiao, Carlos A. Barrero, Rodrigo Vasquez-Del Carpio, E. Premkumar Reddy, Chiara Fecchio, Salim Merali, Alessia Deglincerti, Cheng Fang, Jack Rogers, and Maria L. Maccecchini. "Posiphen Reduces the Levels of Huntingtin Protein through Translation Suppression." Pharmaceutics 13, no. 12 (December 7, 2021): 2109. http://dx.doi.org/10.3390/pharmaceutics13122109.
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Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Posiphen’s safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington’s disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5′-untranslated regions (5′-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.
9
Fortelny, Nikolaus, Christopher M. Overall, Paul Pavlidis, and Gabriela V. Cohen Freue. "Can we predict protein from mRNA levels?" Nature 547, no. 7664 (July 2017): E19—E20. http://dx.doi.org/10.1038/nature22293.
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Watkins, David C., Peter J. Rapiejko, Manuel Ros, Hsien-yu Wang, and Craig C. Malbon. "G-Protein mRNA levels during adipocyte differentiation." Biochemical and Biophysical Research Communications 165, no. 3 (December 1989): 929–34. http://dx.doi.org/10.1016/0006-291x(89)92692-2.
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Udy, Dylan B., and Robert K. Bradley. "Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation." Life Science Alliance 5, no. 3 (December 8, 2021): e202101217. http://dx.doi.org/10.26508/lsa.202101217.
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Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.
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Muller, M., J. Gauley, and John J. Heikkila. "Hydrogen peroxide induces heat shock protein and proto-oncogene mRNA accumulation in Xenopus laevis A6 kidney epithelial cells." Canadian Journal of Physiology and Pharmacology 82, no. 7 (July 1, 2004): 523–29. http://dx.doi.org/10.1139/y04-059.
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In this study, we examined the effect of hydrogen peroxide on the accumulation of various mRNAs encoding heat shock proteins (hsps) and proto-oncogenes in Xenopus A6 kidney epithelial cells. Hydrogen peroxide treatment enhanced the accumulation of hsp90, hsp70, hsp30, c-jun, c-fos, and actin mRNAs with distinct temporal patterns. Although hsp70, c-fos, and c-jun mRNA levels peaked at 1–2 h before declining, hsp30 and hsp90 mRNA levels were maximal at 4–6 h. Other mRNAs, including heat shock cognate hsc70, immunoglobulin binding protein, and ribosomal L8, were unaffected. Treatment of kidney cells with a combination of mild heat shock plus hydrogen peroxide resulted in a synergistic increase in the relative levels of both hsp70 and hsp30 mRNA, but not hsp90, c-fos, c-jun, or actin. This study suggests that analysis of hsp and proto-oncogene mRNA levels may be of value as molecular biomarkers of oxidative stress associated with various disease states and nephrotoxicity in kidney.Key words: Xenopus, kidney, mRNA, heat shock protein, hydrogen peroxide.
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Gygi, Steven P., Yvan Rochon, B. Robert Franza, and Ruedi Aebersold. "Correlation between Protein and mRNA Abundance in Yeast." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1720–30. http://dx.doi.org/10.1128/mcb.19.3.1720.
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ABSTRACT We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeastSaccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
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Laxminarayana, Dama, Islam U. Khan, Nilamadhab Mishra, Irene Olorenshaw, Kjetil Taskén, and Gary M. Kammer. "Diminished Levels of Protein Kinase A RIα and RIβ Transcripts and Proteins in Systemic Lupus Erythematosus T Lymphocytes." Journal of Immunology 162, no. 9 (May 1, 1999): 5639–48. http://dx.doi.org/10.4049/jimmunol.162.9.5639.
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Abstract Deficient type I protein kinase A phosphotransferase activity occurs in the T cells of 80% of subjects with systemic lupus erythematosus (SLE). To investigate the mechanism of this deficient isozyme activity, we hypothesized that reduced amounts of type I regulatory (RI) isoform transcripts, RIα and RIβ, may be associated with a diminution of RIα and/or RIβ protein. Sixteen SLE subjects with a mean (±1 SD) SLE disease activity index of 12.4 ± 7.2 were studied. Controls included 16 normal subjects, six subjects with primary Sjögren’s syndrome (SS), and three subjects with SS/SLE overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells expressed mRNAs for all seven R and catalytic (C) subunit isoforms. Quantification of mRNAs by competitive PCR revealed that the ratio of RIα mRNA to RIβ mRNA in normal T cells was 3.4:1. In SLE T cells there were 20 and 49% decreases in RIα and RIβ mRNAs (RIβ; p = 0.008), respectively, resulting in an RIα:RIβ mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in RIβ mRNA compared with normal controls (p = 0.01). Immunoblotting of normal T cell RIα and RIβ proteins revealed a ratio of RIα:RIβ of 3.2:1. In SLE T cells, there was a 30% decrease in RIα protein (p = 0.002) and a 65% decrease in RIβ protein (p < 0.001), shifting the ratio of RIα:RIβ protein to 6.5:1. T cells from 25% of SLE subjects lacked any detectable RIβ protein. Analysis of several lupus T cell lines demonstrated a persistent deficiency of both proteins, excluding a potential effect of disease activity. In conclusion, reduced expression of RIα and RIβ transcripts is associated with a decrement in RIα and RIβ proteins and may contribute to deficient type I protein kinase A isozyme activity in SLE T cells.
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Chen, Chuan, Xu Zhang, Fei Shang, Haipeng Sun, Baolin Sun, and Ting Xue. "The Staphylococcus aureus Protein-Coding GenegdpSModulatessarSExpression via mRNA-mRNA Interaction." Infection and Immunity 83, no. 8 (June 8, 2015): 3302–10. http://dx.doi.org/10.1128/iai.00159-15.
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Staphylococcus aureusis an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence ofS. aureusis essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein fromStaphylococcus(GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation ofS. aureusNCTC8325. Our previous study showed that the inactivation ofgdpSgenerates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported,sarSis a direct positive regulator ofspa. The decreased transcript levels ofsarSin thegdpSmutant compared with the parental NCTC8325 strain suggest thatgdpSaffectsspathrough interaction withsarS. In this study, site mutation and complementary experiments showed that the translation product ofgdpSwas not involved in the regulation of transcript levels ofsarS. We found thatgdpSfunctioned through direct RNA-RNA base pairing with the 5′ untranslated region (5′UTR) ofsarSmRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis ofsarSin thegdpSmutant showed thatgdpSpositively regulates the mRNA levels ofsarSby contributing to the stabilization ofsarSmRNA, suggesting thatgdpSmRNA may regulatespaexpression in an RNA-dependent pathway.
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Bishola Tshitenge, Tania, and Christine Clayton. "Interactions of the Trypanosoma brucei brucei zinc-finger-domain protein ZC3H28." Parasitology 149, no. 3 (November 2, 2021): 356–70. http://dx.doi.org/10.1017/s003118202100189x.
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AbstractIn Trypanosoma brucei and related Kinetoplastids, regulation of gene expression occurs mostly post-transcriptionally, and RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Trypanosoma brucei ZC3H28 is a 114 KDa cytoplasmic mRNA-binding protein with a single C(x)7C(x)5C(x)sH zinc finger at the C-terminus and numerous proline-, histidine- or glutamine-rich regions. ZC3H28 is essential for normal bloodstream-form trypanosome growth, and when tethered to a reporter mRNA, ZC3H28 increased reporter mRNA and protein levels. Purification of N-terminally tagged ZC3H28 followed by mass spectrometry showed enrichment of ribosomal proteins, various RNA-binding proteins including both poly(A) binding proteins, the translation initiation complex EIF4E4/EIF4G3, and the activator MKT1. Tagged ZC3H28 was preferentially associated with long RNAs that have low complexity sequences in their 3′-untranslated regions; their coding regions also have low ribosome densities. In agreement with the tethering results, after ZC3H28 depletion, the levels of a significant proportion of its bound mRNAs decreased. We suggest that ZC3H28 is implicated in the stabilization of long mRNAs that are poorly translated.
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Ševaljević, L., S. Ivanović-Matić, M. Petrović, M. Glibetić, D. Pantelić, and G. Poznanović. "Regulation of plasma acute-phase protein and albumin levels in the liver of scalded rats." Biochemical Journal 258, no. 3 (March 15, 1989): 663–68. http://dx.doi.org/10.1042/bj2580663.
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At 12 h after scalding of rats a doubling of the hepatocyte nuclear DNA content, which arose from the presence of additional complete genomes and not from amplification of genes coding for the major acute-phase proteins or albumin, was observed. Examination of relative transcription rates per control DNA mass revealed that alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor genes remained constitutive, alpha- and gamma-fibrinogen and haptoglobin genes underwent transcriptional activation for 290 and 339% respectively, whereas the relative transcription rate of albumin decreased to 65% of the control level. Along with these changes, the alpha 1-acid glycoprotein, cysteine-proteinase inhibitor and the fibrinogen mRNA concentrations increased about 500%, haptoglobin mRNA 250%, whereas the albumin mRNA concentration fell to 86% of the control. The regulation of the mRNA levels was assessed by comparing the relative change in transcription rates expressed per control DNA content with the relative changes of mRNA concentrations. We arrived at the conclusion that the concentrations of alpha 1-acid-glycoprotein and cysteine-proteinase-inhibitor mRNAs were predominantly regulated by a post-transcriptional mechanism, albumin mRNA by a transcriptional mechanism, and the fibrinogen and haptoglobin mRNAs by a combination of both. The degree of change of the serum levels of the examined proteins was similar to that of their mRNA concentrations and was the result of the complete use of the available RNA templates in protein synthesis.
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Esser, K. A., and E. C. Hardeman. "Changes in contractile protein mRNA accumulation in response to spaceflight." American Journal of Physiology-Cell Physiology 268, no. 2 (February 1, 1995): C466—C471. http://dx.doi.org/10.1152/ajpcell.1995.268.2.c466.
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Ten rats were exposed to 9 days of zero gravity aboard the National Aeronautics and Space Administration SLS-1 space mission (June 1991). Levels of fast and slow isoform mRNAs from six contractile protein gene families were quantified in the flight soleus and extensor digitorum longus (EDL) muscles. The gene families studied were myosin light chain-1 (MLC-1), myosin light chain-2 (MLC-2), troponin (Tn) T, TnI, TnC, and tropomyosin. In the EDL muscle there was no change in slow mRNA levels with a general increase in fast mRNA levels from 23 to 232%. Changes in slow mRNA levels were seen in the flight soleus muscle with TnCslow and TnTslow levels increasing slightly, and MLC-1slow a, MLC-1slow b, TnIslow, alpha-Tmslow, and MLC-2slow levels decreasing. All fast mRNA levels increased in the flight soleus muscle from 170 to 1,100%. We can conclude that exposure to zero gravity results in 1) a general increase in fast mRNA levels in both fast and slow muscles and 2) differing directional changes in slow mRNA accumulation in the soleus muscle.
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Huang, S., and J. W. Hershey. "Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids." Molecular and Cellular Biology 9, no. 9 (September 1989): 3679–84. http://dx.doi.org/10.1128/mcb.9.9.3679-3684.1989.
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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.
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Huang, S., and J. W. Hershey. "Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids." Molecular and Cellular Biology 9, no. 9 (September 1989): 3679–84. http://dx.doi.org/10.1128/mcb.9.9.3679.
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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.
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Tokuoka, Masafumi, Mizuki Tanaka, Kazuhisa Ono, Shinobu Takagi, Takahiro Shintani, and Katsuya Gomi. "Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression." Applied and Environmental Microbiology 74, no. 21 (September 12, 2008): 6538–46. http://dx.doi.org/10.1128/aem.01354-08.
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ABSTRACT We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3′ rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.
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Kedersha, N., and P. Anderson. "Stress granules: sites of mRNA triage that regulate mRNA stability and translatability." Biochemical Society Transactions 30, no. 6 (November 1, 2002): 963–69. http://dx.doi.org/10.1042/bst0300963.
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Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2α, a key regulatory step in translational initiation. The activation of one or more of the eIF2α kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNAMet, the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNAMet allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2α kinase system, mRNA stability and cellular chaperone levels.
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Roth, Kelly M., Maria K. Wolf, Marie Rossi, and J. Scott Butler. "The Nuclear Exosome Contributes to Autogenous Control of NAB2 mRNA Levels." Molecular and Cellular Biology 25, no. 5 (March 1, 2005): 1577–85. http://dx.doi.org/10.1128/mcb.25.5.1577-1585.2005.
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ABSTRACT The RNA-processing exosome is a complex of riboexonucleases required for 3′-end formation of some noncoding RNAs and for the degradation of mRNAs in eukaryotes. The nuclear form of the exosome functions in an mRNA surveillance pathway that retains and degrades improperly processed precursor mRNAs within the nucleus. We report here that the nuclear exosome controls the level of NAB2 mRNA, encoding the nuclear poly(A)+-RNA-binding protein Nab2p. Mutations affecting the activity of the nuclear, but not the cytoplasmic, exosome cause an increase in the amount of NAB2 mRNA. Cis- and trans-acting mutations that inhibit degradation by the nuclear-exosome subunit Rrp6p result in elevated levels of NAB2 mRNA. Control of NAB2 mRNA levels occurs posttranscriptionally and requires a sequence of 26 consecutive adenosines (A26) in the NAB2 3′ untranslated region, which represses NAB2 3′-end formation and sensitizes the transcript to degradation by Rrp6p. Analysis of NAB2 mRNA levels in a nab2-1 mutant and in the presence of excess Nab2p indicates that Nab2p activity negatively controls NAB2 mRNA levels in an A26- and Rrp6p-dependent manner. These findings suggest a novel regulatory circuit in which the nuclear exosome controls the level of NAB2 mRNA in response to changes in the activity of Nab2 protein.
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LI, Ying, Ulrike MENDE, Carol LEWIS, and Eva J. NEER. "Maintenance of cellular levels of G-proteins: different efficiencies of αs and αo synthesis in GH3 cells." Biochemical Journal 318, no. 3 (September 15, 1996): 1071–77. http://dx.doi.org/10.1042/bj3181071.
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G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein α, β and γ subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, αs and αo, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of αs protein than to produce the same amount of αo protein. Such a situation could arise from a relatively rapid rate of αs protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of αs mRNA compared with αo mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for αs than for αo, yet the rate of αs protein synthesis is only 9 times greater than αo protein synthesis. The degradation rates of the two proteins are similar (13 h for αs and 18 h for αo). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of αs than αo, we asked whether there is a pool of αs mRNA that does not participate in protein synthesis. We found that virtually all αs and αo mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of αs protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.
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Strowski, M. Z., G. Sparmann, H. Weber, F. Fiedler, H. Printz, L. Jonas, B. Göke, and A. C. C. Wagner. "Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins." American Journal of Physiology-Gastrointestinal and Liver Physiology 273, no. 4 (October 1, 1997): G937—G945. http://dx.doi.org/10.1152/ajpgi.1997.273.4.g937.
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We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.
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Sumner, C. J., S. J. Kolb, G. G. Harmison, N. O. Jeffries, K. Schadt, R. S. Finkel, G. Dreyfuss, and K. H. Fischbeck. "SMN mRNA and protein levels in peripheral blood." Neurology 66, no. 7 (February 15, 2006): 1067–73. http://dx.doi.org/10.1212/01.wnl.0000201929.56928.13.
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Background: Clinical trials of drugs that increase SMN protein levels in vitro are currently under way in patients with spinal muscular atrophy.Objective: To develop and validate measures of SMN mRNA and protein in peripheral blood and to establish baseline SMN levels in a cohort of controls, carriers, and patients of known genotype, which could be used to follow response to treatment.Methods: SMN1 and SMN2 gene copy numbers were determined in blood samples collected from 86 subjects. Quantitative reverse transcription PCR was used to measure blood levels of SMN mRNA with and without exon 7. A cell immunoassay was used to measure blood levels of SMN protein.Results: Blood levels of SMN mRNA and protein were measured with high reliability. There was little variation in SMN levels in individual subjects over a 5-week period. Levels of exon 7-containing SMN mRNA and SMN protein correlated with SMN1 and SMN2 gene copy number. With the exception of type I SMA, there was no correlation between SMN levels and disease severity.Conclusion: SMN mRNA and protein levels can be reliably measured in the peripheral blood and used during clinical trials in spinal muscular atrophy, but these levels do not necessarily predict disease severity.
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Stallknecht, B., P. H. Andersen, J. Vinten, L. L. Bendtsen, J. Sibbersen, O. Pedersen, and H. Galbo. "Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes." American Journal of Physiology-Endocrinology and Metabolism 265, no. 1 (July 1, 1993): E128—E134. http://dx.doi.org/10.1152/ajpendo.1993.265.1.e128.
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Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from trained rats. Furthermore, the abundance of the mRNAs for these proteins and glucose transport was measured. Rats were swim-trained for 10 wk, and adipocytes were isolated from epididymal fat pads. The amount of GLUT-4/adipocyte volume unit was significantly higher in trained animals compared with both age- and cell size-matched animals. The amount of GLUT-4 mRNA was also increased by training and it decreased with increasing age. Furthermore, young age as well as training was accompanied by relatively low GLUT-4 protein/mRNA and relatively high overall GLUT-4 efficiency (recruitability and/or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training.
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Castagnola, P., M. Gennari, R. Morello, L. Tonachini, O. Marin, A. Gaggero, and R. Cancedda. "Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein." Journal of Cell Science 110, no. 12 (June 15, 1997): 1351–59. http://dx.doi.org/10.1242/jcs.110.12.1351.
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A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).
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Avignon, A., M. L. Standaert, K. Yamada, H. Mischak, B. Spencer, and R. V. Farese. "Insulin increases mRNA levels of protein kinase C-α and -β in rat adipocytes and protein kinase C-α, -β and -θ in rat skeletal muscle." Biochemical Journal 308, no. 1 (May 15, 1995): 181–87. http://dx.doi.org/10.1042/bj3080181.
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Effects of insulin of levels of mRNA encoding protein kinase C (PKC)-alpha, PKC-beta, PKC-epsilon and PKC-theta were examined by ribonuclease protection assay in primary cultures of rat adipocytes in vitro, and in rat adipose tissue and gastrocnemius muscle in vivo. In all cases, insulin increased the levels of PKC-alpha mRNA and PKC-beta mRNA, and, in muscle, insulin also increased the level of PKC-theta mRNA. PKC-epsilon mRNA levels, on the other hand, were not altered significantly. Insulin also stimulated the apparent translocation of PKC-alpha, -beta, -epsilon and -theta, to the membrane fractions of adipocytes, adipose tissue and gastrocnemius muscles, and, in some instances, total PKC levels were diminished, e.g. PKC-alpha and PKC-beta in cultured adipocytes in vitro and/or whole adipose tissue in vivo, and PKC-alpha and PKC-theta in the gastrocnemius muscle. Thus, insulin-induced increases in PKC mRNA may have been partly compensatory in nature to restore PKC levels following translocation and proteolytic losses. However, much more severe depletion of PKC-alpha and PKC-beta by phorbol ester treatment in cultured rat adipocytes in vitro resulted in, if anything, smaller increases in PKC-alpha mRNA and PKC-beta mRNA, and it therefore appears that insulin effects on PKC mRNA levels were not simply due to decreases in respective PKC levels. In addition, effects of insulin, particularly on PKC-beta mRNA, could not be attributed to increased glucose metabolism, which alone decreased PKC-beta mRNA in cultured adipocytes in vitro. We conclude that insulin-induced translocation and degradation of PKC-alpha, PKC-beta and PKC-theta are attended by selective increases in their mRNAs. This mechanism of increasing mRNA may be important in maintaining PKC levels during the continued action of insulin.
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Lopez, Nelson, John Halladay, William Walter, and Elizabeth A. Craig. "SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes." Journal of Bacteriology 181, no. 10 (May 15, 1999): 3136–43. http://dx.doi.org/10.1128/jb.181.10.3136-3143.1999.
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ABSTRACT Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock theEXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.
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Ishikawa, Tomomoto, Keumsil Hwang, Deborah Lazzarino, and Patricia L. Morris. "Sertoli Cell Expression of Steroidogenic Acute Regulatory Protein-Related Lipid Transfer 1 and 5 Domain-Containing Proteins and Sterol Regulatory Element Binding Protein-1 Are Interleukin-1β Regulated by Activation of c-Jun N-Terminal Kinase and Cyclooxygenase-2 and Cytokine Induction." Endocrinology 146, no. 12 (December 1, 2005): 5100–5111. http://dx.doi.org/10.1210/en.2005-0567.
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In testicular Sertoli cells, IL-1β regulates steroid, lactate, and transferrin secretion; although each influences germ cell development and spermatogenesis, little is known about the signaling mechanisms involved. In other cell types, IL-1β potently induces reactive oxygen species and/or cyclooxygenase-2 (COX-2). In contrast, in Sertoli cells, IL-1β does not generate reactive oxygen species, but rapidly phosphorylates c-Jun-NH2-terminal kinase (JNK), but not p44/42 or p38 MAPK. Phosphorylated JNK stimulates COX-2 activity, mediating the expression of ILs and steroidogenic acute regulatory (StAR)-related (StAR-related lipid transfer protein domain containing) proteins D1 and D5, but not D4. In a time- and dose-dependent manner, IL-1β rapidly increases levels of COX-2 mRNA (2-fold); induction of COX-2 protein (50-fold) requires de novo protein synthesis. Concomitantly, increases in IL-1α, IL-6, and IL-1β mRNAs (1–3 h) are observed. As StAR-related lipid transfer protein domain containing protein 1 (StARD1) mRNA decreases, StARD5 mRNA increases; substantial recovery phase induction of StARD1 mRNA above control is noted (24 h). Inhibition of JNK or COX-2 activities prevents IL-1β induction of IL and StARD5 mRNAs and subsequent increases in StARD1 mRNA (24 h), indicating that these effects depend on the activation of both enzymes. StARD1 and D5 protein levels are significantly altered, consistent with posttranscriptional and posttranslational regulation. IL-1β rapidly decreases levels of precursor and mature sterol regulatory element-binding protein-1, changes not altered by cycloheximide, suggesting coordinate regulation of StARD1 and -D5, but not StARD4, expression. These data demonstrate that JNK and COX-2 activities regulate Sertoli cytokines and particularly START domain-containing proteins, suggesting protective stress responses, including transcription and protein and lipid regulation, within this specialized epithelium.
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Schoolwerth, A. C., P. A. deBoer, A. F. Moorman, and W. H. Lamers. "Changes in mRNAs for enzymes of glutamine metabolism in kidney and liver during ammonium chloride acidosis." American Journal of Physiology-Renal Physiology 267, no. 3 (September 1, 1994): F400—F406. http://dx.doi.org/10.1152/ajprenal.1994.267.3.f400.
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Changes in protein and mRNAs for enzymes of glutamine metabolism were determined in rat kidney cortex at different times after induction of NH4Cl acidosis. After NH4Cl, phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 16-fold by 10 h (P < 0.05) and then returned to control levels by 30 h. In situ hybridization (ISH) showed that PEPCK mRNA was confined to medullary rays; after NH4Cl, expression of PEPCK expanded throughout the cortex, reaching a maximal intensity at 10 h. Phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH) mRNAs increased 8- and 2.6-fold, respectively (both P < 0.05), by 10 h before decreasing; the increased expression was confirmed by ISH. Immunohistochemistry showed that increased PEPCK, PDG, and GDH protein occurred at variable times after the rise in mRNAs. The increase was confined to proximal tubules and was sustained, a finding noted also by Western blot analysis. In contrast, glutamine synthase protein and mRNA, confined to deep cortex and outer medullar, did not change after NH4Cl. These studies reveal striking changes in PEPCK and PDG mRNAs in rat renal cortex during acidosis. The ISH pattern suggested that increased amounts of PEPCK were synthesized in recruited cells which contained little enzyme under physiological conditions. mRNA levels for PEPCK, PDG, and GDH peaked at 10 h before returning to control levels. Despite the decrease in mRNAs, a sustained increase in proteins was noted.
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Yeo, Seung Geun, Sung Jong Lee, Ji Woo Lee, Sujung Oh, and Dong Choon Park. "Levels of endoplasmic reticulum stress-related mRNA in peritoneal fluid of patients with endometriosis or gynaecological cancer." Journal of International Medical Research 49, no. 12 (December 2021): 030006052110653. http://dx.doi.org/10.1177/03000605211065376.
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Objective To compare the levels of endoplasmic reticulum (ER) stress-associated mRNAs and the clinical characteristics of patients with endometriosis or gynaecological cancer. Methods This prospective study obtained intraperitoneal fluid samples from female patients that underwent surgery. The levels of ER stress mRNAs in the peritoneal fluid, including C/EBP-homologous protein (CHOP), X-box binding protein 1 (sXBP1), activating transcription factor 6 (ATF6), immunoglobulin heavy chain-binding protein (BiP), inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), were measured using real-time reverse transcription–polymerase chain reaction in patients with benign disease without endometriosis (control group), with endometriosis or with gynaecological cancer. Results This study enrolled 126 patients: 46 control patients; 47 with endometriosis; and 33 with cancer. The levels of CHOP and BiP mRNA were significantly higher in the control group compared with the cancer group. Levels of sXBP1 and ATF6 mRNA were significantly higher in the cancer group than in the control and endometriosis groups. In the endometriosis group, ATF6 mRNA level was inversely correlated with age and positively correlated with serum cancer antigen 125 levels; and ATF6 and PERK mRNA levels were inversely correlated with parity. Conclusion The levels of ER stress-related mRNAs were related to the pathogenesis of endometriosis and gynaecological cancers.
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Li, Jiawei, Yi Zhang, Cheng Yang, and Ruiming Rong. "Discrepant mRNA and Protein Expression in Immune Cells." Current Genomics 21, no. 8 (December 21, 2020): 560–63. http://dx.doi.org/10.2174/1389202921999200716103758.
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With the development of single-cell mRNA sequencing (scRNA-seq), researchers have attempted to identify new methods for performing in-depth studies of immune cells. However, the discrepancies between the mRNA levels and the levels of surface proteins have confused many researchers. Here, we report a significant and interesting phenomenon in which the mRNA and protein expression levels were mismatched in immune cells. We concluded that scRNA-seq should be combined with other sequencing methods in single-cell studies (e.g., CITE-seq). The simultaneous assessment of both mRNA and protein expression will enhance the precision and credibility of the results.
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Majumder, Mithu, Ibrahim Yaman, Francesca Gaccioli, Vladimir V. Zeenko, Chuanping Wang, Mark G. Caprara, Richard C. Venema, Anton A. Komar, Martin D. Snider, and Maria Hatzoglou. "The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation." Molecular and Cellular Biology 29, no. 10 (March 9, 2009): 2899–912. http://dx.doi.org/10.1128/mcb.01774-08.
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ABSTRACT The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5′ untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.
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Wagner, Eric J., and William F. Marzluff. "ZFP100, a Component of the Active U7 snRNP Limiting for Histone Pre-mRNA Processing, Is Required for Entry into S Phase." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6702–12. http://dx.doi.org/10.1128/mcb.00391-06.
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ABSTRACT Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3′ end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3′ end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3′ end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G1, suggesting that active U7 snRNP is necessary to allow progression through G1 phase to S phase.
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Dykeman, Eric C. "Modelling ribosome kinetics and translational control on dynamic mRNA." PLOS Computational Biology 19, no. 1 (January 23, 2023): e1010870. http://dx.doi.org/10.1371/journal.pcbi.1010870.
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The control of protein synthesis and the overall levels of various proteins in the cell is critical for achieving homoeostasis. Regulation of protein levels can occur at the transcriptional level, where the total number of messenger RNAs in the overall transcriptome are controlled, or at the translational level, where interactions of proteins and ribosomes with the messenger RNA determine protein translational efficiency. Although transcriptional control of mRNA levels is the most commonly used regulatory control mechanism in cells, positive-sense single-stranded RNA viruses often utilise translational control mechanisms to regulate their proteins in the host cell. Here I detail a computational method for stochastically simulating protein synthesis on a dynamic messenger RNA using the Gillespie algorithm, where the mRNA is allowed to co-translationally fold in response to ribosome movement. Applying the model to the test case of the bacteriophage MS2 virus, I show that the models ability to accurately reproduce experimental measurements of coat protein production and translational repression of the viral RNA dependant RNA polymerase at high coat protein concentrations. The computational techniques reported here open up the potential to examine the infection dynamics of a ssRNA virus in a host cell at the level of the genomic RNA, as well as examine general translation control mechanisms present in polycistronic mRNAs.
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Lan, Ping, Wenfeng Li, and Wolfgang Schmidt. "Complementary Proteome and Transcriptome Profiling in Phosphate-deficient Arabidopsis Roots Reveals Multiple Levels of Gene Regulation." Molecular & Cellular Proteomics 11, no. 11 (July 25, 2012): 1156–66. http://dx.doi.org/10.1074/mcp.m112.020461.
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Phosphate (Pi) deficiency impairs plant growth and productivity in many agricultural ecosystems, causing severe reductions in crop yield. To uncover novel aspects in acclimation to Pi starvation, we investigated the correlation between Pi deficiency-induced changes in transcriptome and proteome profiles in Arabidopsis roots. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 13,298 proteins and 24,591 transcripts, subsets of 356 proteins and 3106 mRNAs were differentially expressed during Pi deficiency. Most dramatic changes were noticed for genes involved in Pi acquisition and in processes that either liberate Pi or bypass Pi/ATP-consuming metabolic steps, for example during membrane lipid remodeling and glycolytic carbon flux. The concordance between the abundance of mRNA and its encoded protein was generally high for highly up-regulated genes, but the analysis also revealed numerous discordant changes in mRNA/protein pairs, indicative of divergent regulation of transcription and post-transcriptional processes. In particular, a decreased abundance of proteins upon Pi deficiency was not closely correlated with changes in the corresponding mRNAs. In several cases, up-regulation of gene activity was observed solely at the protein level, adding novel aspects to key processes in the adaptation to Pi deficiency. We conclude that integrated measurement and interpretation of changes in protein and transcript abundance are mandatory for generating a complete inventory of the components that are critical in the response to environmental stimuli.
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Laye, Matthew J., Thomas P. J. Solomon, Kristian Karstoft, Karin K. Pedersen, Susanne D. Nielsen, and Bente K. Pedersen. "Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event." Journal of Applied Physiology 112, no. 5 (March 1, 2012): 773–81. http://dx.doi.org/10.1152/japplphysiol.00997.2011.
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Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects ( n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 ( P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4+ or CD8+ cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.
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Eble, D. M., and F. G. Spinale. "Contractile and cytoskeletal content, structure, and mRNA levels with tachycardia-induced cardiomyopathy." American Journal of Physiology-Heart and Circulatory Physiology 268, no. 6 (June 1, 1995): H2426—H2439. http://dx.doi.org/10.1152/ajpheart.1995.268.6.h2426.
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Chronic supraventricular tachycardia (SVT)-induced cardiomyopathy is associated with left ventricular (LV) dilatation, increased wall stress, neurohormonal activation, and no change in LV mass. To determine mechanisms for changes in LV geometry and function with SVT cardiomyopathy, LV and myocyte function, contractile protein content and mRNA levels, and cytoskeletal protein structure and mRNA levels were examined in 12 pigs with SVT cardiomyopathy (paced at 240 beats/min for 3 wk) and in 12 controls. With SVT cardiomyopathy, LV fractional shortening fell by 61%, and end-diastolic dimension increased by 42%, with no change in LV mass-to-body weight ratio (3.36 +/- 0.15 vs. 3.14 +/- 0.13 g/kg). Myocyte contractile function was reduced by 33%, myocyte length was increased by 28%, and cross-sectional area was decreased by 19% with SVT cardiomyopathy. Total protein, myosin heavy chain (MHC), and actin appeared unchanged with SVT cardiomyopathy at the LV or myocyte level. Moreover, there was no change in mRNA levels for MHC (0.57 +/- 0.05 vs. 0.59 +/- 0.09 mRNAOD/rRNAOD) or cardiac alpha-actin (0.58 +/- 0.08 vs. 0.58 +/- 0.04 mRNAOD/rRNAOD) with SVT cardiomyopathy. In contrast, mRNA levels for specific cytoskeletal proteins were significantly increased with SVT cardiomyopathy, and immunofluorescent localization of contractile and cytoskeletal proteins in isolated myocytes revealed alterations in cytoskeletal architecture. Thus changes in LV and myocyte geometry with SVT cardiomyopathy were associated with no change in contractile protein content or mRNA at the chamber or myocyte level. Furthermore, increased cytoskeletal protein abundance and mRNA and reorientation of cardiocyte cytoarchitecture may have contributed to the LV and myocyte remodeling with SVT cardiomyopathy.
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Yeh, Kwo-Yih, Mary Yeh, and Jonathan Glass. "Expression of intestinal brush-border membrane hydrolases and ferritin after segmental ischemia-reperfusion in rats." American Journal of Physiology-Gastrointestinal and Liver Physiology 275, no. 3 (September 1, 1998): G572—G583. http://dx.doi.org/10.1152/ajpgi.1998.275.3.g572.
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Jejunal expression of three brush-border membrane (BBM) enzymes, intestinal alkaline phosphatase (IAP), lactose-phlorizin hydrolase (LPH), and sucrase-isomaltase (SI), and a cytosolic protein, ferritin (Ft), was investigated after transient segmental ischemia-reperfusion (I/R). I/R reduced mucosal IAP, LPH, and SI mRNAs to 36%, 11%, and 38% of normal jejunal levels after 3 h of reperfusion and to 22%, 8%, and 51% of normal jejunal levels after 6 h of reperfusion, respectively. Intriguingly, in the internal control jejunum IAP and LPH mRNAs also decreased significantly. LPH and SI mRNA rapidly recovered to levels significantly higher than those of normal jejunum at 12 h, whereas IAP mRNA levels did not recover until 48 h. Enzyme activity paralleled changes in mRNA levels in the ischemic reperfused jejunum. Electrophoretic mobility shift assays showed that I/R significantly increased SI footprinting 1 (SIF1) binding activity. The mobility of one of the DNA-protein complexes was further retarded in the presence of anti-Cdx-2 antibody, suggesting that either Cdx-2 or a related protein was interacting with the SIF1 sequences. Similar to BBM enzymes, cytosolic Ft mRNA and protein were significantly decreased at 3 and 6 h after I/R. By 12 h, Ft mRNA, but not Ft protein, had increased to higher than normal levels. We conclude that a rapid recovery of BBM mRNAs and enzymes occurs in regenerating mucosa after upper villus damage. The increase of SIF1 binding protein activity after I/R may enhance SI, and perhaps LPH, gene transcription. The expression of Ft is regulated at both pretranslational and translational levels.
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Milland, J., A. Tsykin, T. Thomas, A. R. Aldred, T. Cole, and G. Schreiber. "Gene expression in regenerating and acute-phase rat liver." American Journal of Physiology-Gastrointestinal and Liver Physiology 259, no. 3 (September 1, 1990): G340—G347. http://dx.doi.org/10.1152/ajpgi.1990.259.3.g340.
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The integration of growth and the acute-phase response is investigated by comparing the mRNA levels in rat liver during acute inflammation with those after partial hepatectomy. Northern analysis is carried out for the mRNAs for thiostatin, alpha 2-macroglobulin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor subunit 1, haptoglobin, ceruloplasmin, transferrin, vitamin D-binding protein, alpha 1-acid glycoprotein, beta-fibrinogen, apolipoproteins A-IV and E, albumin, transthyretin, alpha 2-HS-glycoprotein, retinol-binding protein, beta-tubulin, c-myc protooncogene, glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, ornithine transcarbamylase, and alcohol dehydrogenase. The acute-phase response dominates during the first 18 h. Changes in mRNA levels related to growth of the liver become important thereafter, and the capacity for an acute-phase response of plasma protein synthesis becomes greatly reduced. The early increase in the level of ceruloplasmin mRNA observed during inflammation is abolished during regeneration, and that of vitamin D-binding protein mRNA is converted into a decrease. The mRNAs levels of glyceraldehyde-3-phosphate dehydrogenase increase, and those for phosphoenolpyruvate carboxykinase decrease during regeneration. Ornithine transcarbamylase mRNA levels are found to exhibit negative acute-phase regulation. The pattern of transcriptional regulation is similar during inflammation and regeneration.
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Kawase, Atsushi, Akira Kazaoka, Rei Yamamoto, Risa Minakata, Hiroaki Shimada, and Masahiro Iwaki. "Changes in Transporters and Metabolizing Enzymes in the Livers of Rats with Bile Duct Ligation." Journal of Pharmacy & Pharmaceutical Sciences 22 (September 19, 2019): 457–65. http://dx.doi.org/10.18433/jpps30637.
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Purpose: Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. Methods: Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Results: Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. Conclusions: This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.
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Hu, S., Y. Li, J. Wang, Y. Xie, K. Tjon, L. Wolinsky, R. R. O. Loo, J. A. Loo, and D. T. Wong. "Human Saliva Proteome and Transcriptome." Journal of Dental Research 85, no. 12 (December 2006): 1129–33. http://dx.doi.org/10.1177/154405910608501212.
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This paper tests the hypothesis that salivary proteins and their counterpart mRNAs co-exist in human whole saliva. Global profiling of human saliva proteomes and transcriptomes by mass spectrometry (MS) and expression microarray technologies, respectively, revealed many similarities between saliva proteins and mRNAs. Of the function-known proteins identified in saliva, from 61 to 70% were also found present as mRNA transcripts. For genes not detected at both protein and mRNA levels, we made further efforts to determine if the counterpart is present. Of 19 selected genes detected only at the protein level, the mRNAs of 13 (68%) genes were found in saliva by RT-PCR. In contrast, of many mRNAs detected only by microarrays, their protein products were found in saliva, as reported previously by other investigators. The saliva transcriptome may provide preliminary insights into the boundary of the saliva proteome.
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Ros, M., D. C. Watkins, P. J. Rapiejko, and C. C. Malbon. "Glucocorticoids modulate mRNA levels for G-protein β-subunits." Biochemical Journal 260, no. 1 (May 15, 1989): 271–75. http://dx.doi.org/10.1042/bj2600271.
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Adrenalectomy decreases, whereas glucocorticoid treatment increases, the steady-state levels of G-protein beta-subunits (G beta) in rat fat-cells. A DNA-excess solution-hybridization assay was established to define the steady-state mRNA levels for G beta [5.8 +/- 0.4 amol/micrograms of RNA (n = 5) in control fat-cells]. G beta mRNA levels decrease by 20% after adrenalectomy; dexamethasone treatment reverses the decline. Dexamethasone treatment itself increases G beta mRNA levels by 50%.
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Duffy, Carol, Ekaette F. Mbong, and Joel D. Baines. "VP22 of Herpes Simplex Virus 1 Promotes Protein Synthesis at Late Times in Infection and Accumulation of a Subset of Viral mRNAs at Early Times in Infection." Journal of Virology 83, no. 2 (November 5, 2008): 1009–17. http://dx.doi.org/10.1128/jvi.02245-07.
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ABSTRACT VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus 1 (HSV-1) tegument. In the present study we show VP22 is required for optimal protein synthesis at late times in infection. Specifically, in the absence of VP22, viral proteins accumulated to wild-type levels until ∼6 h postinfection. At that time, ongoing synthesis of most viral proteins dramatically decreased in the absence of VP22, whereas protein stability was not affected. Of the individual proteins we assayed, VP22 was required for optimal synthesis of the late viral proteins gE and gD and the immediate-early protein ICP0 but did not have discernible effects on accumulation of the immediate-early proteins ICP4 or ICP27. In addition, we found VP22 is required for the accumulation of a subset of mRNAs to wild-type levels at early, but not late, times in infection. Specifically, the presence of VP22 enhanced the accumulation of gE and gD mRNAs until ∼9 h postinfection, but it had no discernible effect at later times in infection. Also, VP22 did not significantly affect ICP0 mRNA at any time in infection. Thus, the protein synthesis and mRNA phenotypes observed with the UL49-null virus are separable with regard to both timing during infection and the genes affected and suggest separate roles for VP22 in enhancing the accumulation of viral proteins and mRNAs. Finally, we show that VP22's effects on protein synthesis and mRNA accumulation occur independently of mutations in genes encoding the VP22-interacting partners VP16 and vhs.
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Warringer, Jonas, Malin Hult, Sergi Regot, Francesc Posas, and Per Sunnerhagen. "The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock." Molecular Biology of the Cell 21, no. 17 (September 2010): 3080–92. http://dx.doi.org/10.1091/mbc.e10-01-0006.
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Cellular responses to environmental changes occur on different levels. We investigated the translational response of yeast cells after mild hyperosmotic shock by isolating mRNA associated with multiple ribosomes (polysomes) followed by array analysis. Globally, recruitment of preexisting mRNAs to ribosomes (translational response) is faster than the transcriptional response. Specific functional groups of mRNAs are recruited to ribosomes without any corresponding increase in total mRNA. Among mRNAs under strong translational up-regulation upon shock, transcripts encoding membrane-bound proteins including hexose transporters were enriched. Similarly, numerous mRNAs encoding cytoplasmic ribosomal proteins run counter to the overall trend of down-regulation and are instead translationally mobilized late in the response. Surprisingly, certain transcriptionally induced mRNAs were excluded from ribosomal association after shock. Importantly, we verify, using constructs with intact 5′ and 3′ untranslated regions, that the observed changes in polysomal mRNA are reflected in protein levels, including cases with only translational up-regulation. Interestingly, the translational regulation of the most highly osmostress-regulated mRNAs was more strongly dependent on the stress-activated protein kinases Hog1 and Rck2 than the transcriptional regulation. Our results show the importance of translational control for fine tuning of the adaptive responses.
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Heikkila, J. J., N. Ovsenek, and P. Krone. "Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos." Biochemistry and Cell Biology 65, no. 2 (February 1, 1987): 87–94. http://dx.doi.org/10.1139/o87-013.
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Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33–35 °C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27–37 °C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 °C, with relatively greater levels at 30–35 °C and lower levels at 37 °C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27–35 °C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33–35 °C induced maximum relative levels of hsp 70 mRNA within 1–1.5 h, while at 30 and 27 °C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively. Finally, placement of Xenopus neurulae at 22 °C after a 1-h heat shock at 33 °C produced an initial decrease in hsp 70 mRNA within 15–30 min, followed by a transient increase in hsp 70 mRNA at 1–2 h before decaying to background levels by 7 h.
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Hartmann, Claudia, Corinna Benz, Stefanie Brems, Louise Ellis, Van-Duc Luu, Mhairi Stewart, Iván D'Orso, et al. "Small Trypanosome RNA-Binding Proteins TbUBP1 and TbUBP2 Influence Expression of F-Box Protein mRNAs in Bloodstream Trypanosomes." Eukaryotic Cell 6, no. 11 (September 1, 2007): 1964–78. http://dx.doi.org/10.1128/ec.00279-07.
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ABSTRACT In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3′-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, TbUBP1 and TbUBP2, containing a single RNA recognition motif, in trypanosome gene expression. TbUBP1 and TbUBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of TbUBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3′-untranslated region and involved stabilization of the mRNA. Depletion of TbUBP2 and TbUBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to TbUBP2 or TbUBP1, with some preference for CFB1. We suggest that TbUBP1 and TbUBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.
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Dominguez, J. H., C. C. Hale, and M. Qulali. "Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters." American Journal of Physiology-Renal Physiology 270, no. 2 (February 1, 1996): F245—F253. http://dx.doi.org/10.1152/ajprenal.1996.270.2.f245.
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Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.