Experimental nutrition. Synthesis of ribosomal and messenger RNA in fed and fasted rats.
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Animals↗
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Alternatives to 35S as a label for the differential display of eukaryotic messenger RNA.
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Arabidopsis↗
Cell-free translation of paramyxovirus messenger RNA.
Polypeptides corresponding in electrophoretic mobility to virion polypeptides 1, 3, and 5 were made in a reticulocyte cell-free system to which 18S RNA from Sendai virus-infected cells was added. Immune precipitation was used to select relevant polypeptides from endogenous products. The cell-free product corresponding to virion polypeptide 3 (the nucleocapsid structure unit) was the most abundant; its tryptic peptides comigrated electrophoretically with tryptic peptides of polypeptide 3 isolated from virions. Other sedimenting classes of RNA from infected cells were tested; only the 28S fraction showed slight activity. Virion 50S RNA was inactive. These findings support the hypothesis that complementary RNA transcripts of paramyxovirion RNA are the templates for viral proteins.
Animals↗
Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells.
RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.
Animals↗
Synthesis of albumin with exogenous mouse-liver messenger RNA in a homologous cell-free system.
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Albumins↗
Endogenous viral enzymes involved in messenger RNA production.
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DNA-Directed RNA Polymerases↗
Linking transcriptional elongation and messenger RNA export to metastatic breast cancers.
The biochemical pathways that are disrupted in the genesis of sporadic breast cancers remain unclear. Moreover, the present prognosticating markers used to determine the prognosis of node-negative patient leads to probabilistic results, and the eventual clinical course is far from certain. Here we identified the human TREX complex, a multiprotein complex that links transcription elongation to mRNA transport, as culprit of aggressive human breast cancers. We show that whereas p84N5 (called hTREX84) is expressed at very low levels in normal breast epithelial cells, it is highly expressed in breast tumors. Importantly, hTREX84 expression correlates with tumor size and the metastatic state of the tumor progression. Reduction of hTREX84 levels in breast cancer cell lines by small interfering RNA result in inhibition of cellular proliferation and abrogation of mRNA export. These results not only identify hTREX84 as a prognosticator of breast cancer but also delineate human TREX complex as a target for therapeutic drugs against breast cancer.
Adult↗
CaSm-mediated cellular transformation is associated with altered gene expression and messenger RNA stability.
CaSm (cancer-associated Sm-like) was originally identified based on elevated expression in pancreatic cancer and in several cancer-derived cell lines. CaSm encodes a 133 amino acid protein that contains two Sm motifs found in the common small nuclear RNA proteins and the LSm (like-Sm) family of proteins. Compared with normal human prostate tissue and primary prostate epithelial cells, some primary prostate tumors and prostate cancer-derived cell lines have elevated CaSm expression. Expression of antisense CaSm RNA in DU145 cells results in reduced CaSm protein levels and less transformed phenotype, measured by anchorage-independent growth in vitro and tumor formation in severe combined immunodeficient mice in vivo. Additional data shows that adenoviral delivery of antisense CaSm inhibits the growth of prostate cancer cell lines by altering cell cycle progression, and is associated with reduced expression of cyclin B1 and CDK1 proteins. Consistent with failure of antisense-treated cells to enter mitosis, microarray analysis identified altered expression of NEK2 and nucleophosmin/B23. Although the mechanisms by which CaSm contributes to neoplastic transformation and cellular proliferation are unknown, it has been shown that the yeast homologue (spb8/LSm1) of CaSm is required for 5' to 3' degradation of specific mRNAs. We provide data consistent with a similar role for CaSm in human cells, supporting the hypothesis that elevated CaSm expression observed in cancer leads to destabilization of multiple gene transcripts, contributing to the mutator phenotype of cancer cells.
Adenoviridae↗
Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.
The purpose of this study was to compare the detection of human papillomavirus (HPV) DNA with detection of mRNA. The study included 4,136 women >30 years of age. E6/E7 mRNA expression from the carcinogenic HPV types 16, 18, 31, 33, and 45 was detected by the PreTect HPV-Proofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by type-specific PCR. A total of 4.0% had an abnormal cytologic diagnosis, 3.0% were positive by PreTect HPV-Proofer, 4.4% by type-specific PCR, and 10.4% by consensus PCR. For detection of HPV in high-grade squamous intraepithelial lesion (HSIL), no significant difference was observed between PreTect HPV-Proofer and consensus PCR. For women with a cytologic normal, atypical squamous cell of uncertain significance, and low-grade SIL diagnosis, the detection rate of HPV was significantly higher by Gp5+/6+ consensus PCR (P < 0.005) than by PreTect HPV-Proofer. Histology confirmed 14 of 23 cytologic HSIL as cervical intraepithelial neoplasia grade >2. Of these women, PreTect HPV-Proofer and type-specific PCR detected 12, whereas consensus PCR detected 13. In conclusion, for HSIL, detection of E6/E7 transcripts from HPV types 16, 18, 31, 33, and 45 are present to the same degree as DNA detected by consensus PCR. Equally important, only a small proportion of the HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or low-grade SIL diagnosis had a detectable mRNA expression. HPV E6/E7 mRNA detection by PreTect HPV-Proofer represents a new promising test as an adjunct to cytology.
Adult↗
Quantitative assessment of telomerase activity and human telomerase reverse transcriptase messenger RNA levels in pancreatic juice samples for the diagnosis of pancreatic cancer.
Measurement of telomerase activity is a promising diagnostic tool for pancreatic cancer. Detection of mRNA for human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is also a diagnostic candidate. In the present study, we developed a telomeric repeat amplification protocol assay with real-time PCR and a protocol for quantification of hTERT mRNA with real-time PCR. To evaluate the feasibility of these methods for diagnosis of pancreatic cancer, we measured telomerase activity and hTERT expression in pancreatic cancer cell lines, pancreatic tissues, and pancreatic juice samples from patients with different pancreatic diseases. There were significant correlations between telomerase activity and hTERT expression in cell lines, tissues, and juice samples. The levels of telomerase activity and hTERT expression were significantly higher in tumoral tissues than in nontumoral tissues. In pancreatic juice specimens, some carcinoma samples showed remarkably high expression of hTERT. However, there were no significant differences in hTERT expression between patients with carcinoma and those with benign diseases, although significant differences in telomerase activity were observed. Our present results suggest that the combined assessment of hTERT and telomerase activities in pancreatic juice provides a potent diagnostic method for pancreatic cancer.
Adenocarcinoma↗
Carcinoembryonic antigen messenger RNA expression in blood predicts recurrence in esophageal cancer.
PURPOSE: The clinical significance of isolated tumor cells (ITC) in blood has not been clearly established, particularly during follow-up in cancer patients. We conducted a longitudinal analysis of the relationship between ITC in blood during follow-up and clinicopathologic findings in patients with esophageal squamous cell carcinoma. EXPERIMENTAL DESIGN: Blood samples obtained from 106 patients were examined by real-time RT-PCR assay targeting carcinoembryonic antigen (CEA) mRNA. Follow-up examination every 3 months after surgery included testing for CEA mRNA and tumor markers, as well as imaging. RESULTS: Thirty-nine (36.8%) patients were positive for CEA mRNA expression. CEA mRNA positivity significantly correlated with tumor depth, lymph node metastasis, stage, and venous invasion. Recurrent disease was found in 34 of 106 (32.1%) cases. CEA mRNA was found in 28 (76.5%) patients experiencing relapse. Of these 28 patients, the number positive of CEA mRNA before detection by imaging, at the same time of detection by imaging, and after detection by imaging was 18 (52.9%), 8 (23.5%), and 2 (5.9%), respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for CEA mRNA were higher than those for serum CEA or squamous cell carcinoma. Patients positive for CEA mRNA experienced significantly shorter disease-free interval than those with negative CEA mRNA (P < 0.001). According to multivariate analysis, CEA mRNA positivity was an independent factor for disease-free interval. CONCLUSIONS: Examination of CEA mRNA in peripheral blood during follow-up is useful for early detection of occult recurrence. We believe that CEA mRNA in blood will be a new marker for recurrence in esophageal squamous cell carcinoma.
Adult↗
Identification of messenger RNA for nucleocapsid protein of Lassa virus in infected cells.
Single-stranded RNAs from Lassa virus-infected cells were fractionated by affinity chromatography on an oligo(dT)-cellulose column as well as by sucrose gradient centrifugation. Fractionated RNAs were translated in rabbit reticulocyte lysates, and translation products were precipitated with monoclonal antibodies to the nucleocapsid protein of Lassa virus. It has been shown that mRNA for the nucleocapsid protein is 15-16S and the RNA does not contain long if any poly(A) sequences.
Animals↗
Full-length messenger RNA sequences greatly improve genome annotation.
BACKGROUND: Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism. RESULTS: Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation. CONCLUSIONS: Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.
Alternative Splicing↗
Culture at high density increases phosphoenolpyruvate carboxykinase messenger RNA in H4IIEC3 hepatoma cells.
We report that the concentration of phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 5- to 10-fold when H4IIEC3 rat hepatoma cells were cultured at high compared to low density. The magnitude and direction of this response were mRNA specific, as the mRNAs encoding tyrosine aminotransferase and albumin increased approximately 20%, whereas the mRNAs encoding beta-actin and alpha-tubulin decreased 40% and 20%, respectively. Paracrine or autocrine mechanisms were not responsible for the density effect, since conditioned medium or frequent medium changes had only a modest effect on the abundance of PEPCK mRNA. Culture of H4IIEC3 cells at low density or on collagen promoted a flattened morphology and low PEPCK mRNA levels. At high density, cells assumed a cuboidal shape on both plastic and collagen and expressed high PEPCK mRNA levels. Induction of PEPCK mRNA by high density culture did not involve increased intracellular cAMP, since treatment with 8-(4-chlorophenylthio)-cAMP was synergistic with density. High cell density increased PEPCK run-on transcription approximately 3-fold, while PEPCK mRNA increased more than 6-fold. These observations suggest that high culture density increases PEPCK mRNA by increasing its transcription and possibly stabilizing PEPCK mRNA. The response could be coupled to the regulation of cell shape, as a close relationship between cell shape and gene expression has previously been shown to be important in the development and maintenance of tissues and organs. The PEPCK gene in H4IIEC3 cells could provide a useful model in which to study the poorly understood mechanisms involved in coordinating form and function.
Animals↗
Posttranscriptional regulation of albumin and alpha-fetoprotein messenger RNA by transforming growth factor-beta 1 requires de novo RNA and protein synthesis.
Transforming growth factor-beta (TGF beta) has been implicated in the regulation of hepatocyte function. We have examined TGF beta 1 regulation of albumin and alpha-fetoprotein (AFP) mRNA levels in a well differentiated mouse hepatoma cell line (BWTG3). TGF beta 1 reversibly decreased steady state mRNA levels of both albumin and AFP. By nuclear run-on assays, we found that TGF beta 1 caused no significant change in transcription rates for albumin or AFP. Pretreatment with actinomycin-D prevented the TGF beta 1-induced decrease in albumin and AFP mRNA levels. Also, if cells were treated with actinomycin-D after a 12-h exposure to TGF beta 1, actinomycin-D abrogated the further decrease in albumin and AFP mRNA levels that occurred after treatment with TGF beta 1 alone. Cycloheximide pretreatment blocked the TGF beta 1-induced decrease in albumin and AFP mRNA levels. TGF beta 1 altered neither the rate of BWTG3 cell growth nor the levels of mRNA for the growth-associated protooncogene c-myc. These data suggest that TGF beta 1 has regulatory effects on specific hepatocyte functions that are independent of growth regulatory effects. The decrease in albumin and AFP mRNAs caused by TGF beta 1 is posttranscriptional and dependent upon de novo RNA and protein synthesis.
Albumins↗
The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA.
The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.
Animals↗
Homologous up-regulation of the gonadotropin-releasing hormone receptor in alpha T3-1 cells is associated with unchanged receptor messenger RNA (mRNA) levels and altered mRNA activity.
In primary cultures of rat pituitary, GnRH receptor has been reported to up-regulate when exposed to GnRH. The molecular basis of this regulation could, in theory, involve modulation of gene transcription, RNA processing or stability, translation, and/or receptor degradation. The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, alpha T3-1 cells, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on alpha T3-1 cell membranes. To correlate changes in receptor number and measurements of biosynthetic activity, GnRH receptor mRNA levels were quantitated by solution hybridization/RNase protection assay and Northern blot analysis, and the capacity of RNA from treated cells to direct the synthesis of new receptors (mRNA activity) was evaluated using a Xenopus oocyte-based bioassay. GnRH agonist radioligand binding assay results showed that exposure of alpha T3-1 cells to 10(-10) or 10(-8) M GnRH for 20 min induced an approximately 50% increase in the number of GnRH receptors, similar to previously reported results in rat pituitary primary culture. Despite the increase in receptor number, however, cytosolic and total GnRH receptor mRNA levels assayed by solution hybridization/RNase protection assay with GnRH receptor cRNA probes and by Northern blot analysis were not altered. In contrast to the unchanging mRNA levels, the measurements of mRNA activity paralleled the changes observed at the binding level. alpha T3-1 RNA from treated and control cells were injected into Xenopus oocytes, and the GnRH-induced Cl- current was quantified 48 h later by voltage clamp recording of the response to GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Regulation of endogenous thyrotropin-releasing hormone receptor messenger RNA in GH4C1 cells: roles of protein and RNA synthesis.
In previous studies on the regulation of the endogenous TRH receptor (TRHR) in GH4C1 cells, we have shown that the synthetic glucocorticoid dexamethasone (Dex) increased TRHR abundance on the plasma membrane of these cells as well as increasing the concentration of TRHR mRNA. In the present investigation, we determined whether the action of Dex on TRHR mRNA was direct or whether it involved the synthesis of intermediary protein(s). We found that the protein synthesis inhibitors cycloheximide or anisomycin alone enhanced TRHR mRNA accumulation and, in the presence of Dex, caused a 10- to 20-fold superinduction of TRHR mRNA. The superinduction required inhibition of protein synthesis. Results of nuclear run-on assays showed that superinduction was accompanied by a large increase in transcription of TRHR mRNA. Experiments designed to examine the stability of TRHR mRNA transcripts revealed that inhibitors of RNA synthesis (actinomycin D and dichlorobenzimidazole riboside) caused an increase in endogenous TRHR mRNA concentrations by themselves, complicating their use to estimate TRHR mRNA half-life. We conclude that Dex stimulates transcription of endogenous TRHR mRNA by a mechanism that does not require enhanced synthesis of new proteins. In fact, ongoing protein synthesis reduces steady state TRHR mRNA concentrations consistent with a role for a labile protein(s) in suppressing TRHR gene transcription; an additional action of a labile protein to enhance TRHR mRNA degradation is also possible.
Animals↗