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At least 919 records · Page 51Linked to original sources
PubMed2006

Messenger RNA turnover and its regulation in herpesviral infection.

The ability to regulate cellular gene expression is a key aspect of the lifecycles of a diverse array of viruses. In fact, viral infection often results in a global shutoff of host cellular gene expression; such inhibition serves not only to ensure maximal viral gene expression without competition from the host for essential machinery and substrates but also aids in evasion of immune responses detrimental to successful viral replication and dissemination. Within the herpesvirus family, host shutoff is a prominent feature of both the alpha- and gamma-herpesviruses. Intriguingly, while both classes of herpesviruses block cellular gene expression by inducing decay of messenger RNAs, the viral factors responsible for this phenotype as well as the mechanisms by which it is achieved are quite distinct. However, data suggest that the host shutoff functions of alpha- and gamma-herpesviruses are likely achieved both through the activity of virally encoded nucleases as well as via modulation of cellular RNA degradation pathways. This review highlights the processes governing normal cellular messenger RNA decay and then details the mechanisms by which herpesviruses promote accelerated RNA turnover. Parallels between the viral and cellular degradation systems as well as the known interactions between viral host shutoff factors and the cellular RNA turnover machinery are highlighted.

Animals↗
PubMed2002

Nonsense-mediated decay: assaying for effects on selenoprotein mRNAs.

If the abundance of a particular selenoprotein mRNA is reduced during selenium deprivation, then the mRNA is likely to be a natural substrate for NMD. One assay for NMD involves changing the TGA Sec codon(s) to either a TGC cysteine codon or a TAA nonsense codon. If selenium deprivation elicits NMD and has no other effect on selenoprotein gene expression, then, regardless of selenium concentration, the level of UGC-containing mRNA should be most abundant, the level of UGA-containing mRNA should be intermediate in abundance, and the level of UAA-containing mRNA should be least abundant. Furthermore, the level of UGA-containing mRNA should be decreased by a decrease in selenium concentration, while the levels of UGC- and UAA-containing mRNAs should be unaffected by selenium concentration. A different assay for NMD involves coexpression of the particular selenoprotein gene and a vector expressing a dominant-negative version of hUpf1p. This assay is simpler and more versatile than the first assay because it can be used to assay any cellular gene in situ provided the cells can be stably transfected with the hUpf1p expression vector.

Animals↗
PubMed2001

Functional link between the mammalian exosome and mRNA decapping.

Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3' to 5' exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.

Animals↗
PubMed1999

Analysis of the viability of freezer stored serum samples for hepatitis C virus RNA analysis by the SUPERQUANT method: results of a 16 year retrospective study.

Prior to the discovery of the hepatitis C virus (HCV), virological analysis of serum from patients with non-A non-B hepatitis was not possible. Since the finding that HCV is the causative agent of most non-A non-B hepatitis, several reliable methodologies have been developed that allow for quantification of HCV RNA. To determine the viability of stored serum samples for HCV RNA analysis. 256 samples were examined for HCV RNA using a multi-cycle RT-PCR assay. All samples were stored unopened in a -70 degree C freezer until the time of testing. Collection years ranged from 1981 to 1995. To examine the integrity of stored serum samples, the distribution of quantitative HCV RNA values for each year was compared: year-to-year; and, to the distribution of HCV RNA concentrations from 1510 chronic HCV patients determined by the same assay in 1996 and 1997. Pairwise year-to-year analysis revealed that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). Likewise, comparison of the stored samples to the 1510 fresh samples demonstrated that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). The results demonstrate a method for determination of the integrity of stored serum samples from chronic HCV patients. The mechanism of RNA degradation is unknown but it is most likely to be due to poor sample collection procedures in place prior to 1991.

Adult↗
PubMed2000

Role of TNFalpha in regulation of myeloperoxidase expression in irradiated HL60 promyelocytic cells.

Irradiation increases the generation of reactive oxygen intermediates, including hydrogen peroxide (H(2)O(2)). Myeloperoxidase (MPO), a heme-containing glycoprotein located in the primary granules of polymorphonuclear leukocytes and monocytes, reacts with H(2)O(2) and halide ion and produces a more potent microbicidal oxidant, hypochlorous acid (HOCl). Human HL60 promyelocytes constitutively had high levels of MPO protein and mRNA. Irradiation decreased the levels of MPO transcripts; the decrease in MPO transcripts by irradiation occurred in an almost dose-dependent manner. HL60 cells produce tumor necrosis factor alpha (TNFalpha), and irradiation markedly increased the TNFalpha production in these cells; in turn, TNFalpha decreased the levels of MPO transcripts in these cells. Furthermore, treatment of cells with anti-TNFalpha antibody blocked the reduction of MPO by irradiation. We also found that irradiation decreased the levels of the MPO mRNA with concomitant increased levels of TNFalpha mRNA in differentiation-induced HL60 cells and human THP-1 monocytic cells. Irradiation reduced the rate of MPO transcription but had only a slight effect on the half-life of MPO mRNA in HL60 cells. Our results suggest that irradiation reduces the steady-state levels of MPO mRNA mainly at transcriptional level and the endogenous production of TNFalpha is required for the reduction by irradiation in HL60 cells.

Cell Differentiation↗
PubMed2002

The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter.

In order to develop a highly efficient mammalian expression vector, we constructed a vector by the combination of the murine cytomegalovirus (MCMV) immediate early (IE) promoter and the human elongation factor one alpha (EF-1 alpha) first intron. The MCMV IE promoter was several fold stronger than the human cytomegalovirus (HCMV) immediate early (IE) promoter and the human elongation factor one alpha (EF-1 alpha) promoter in various mammalian cell lines such as NIH3T3, Neuro-2a, 293T or HT1080 and was only slightly weaker than the HCMV or the EF-1 alpha promoter in HeLa and CHO cell lines. We inserted the first intron of the human EF-1 alpha gene behind the MCMV and the HCMV promoter to enhance the gene expression through increasing RNA transcription and/or RNA stability. The insertion of the human EF-1 alpha first intron markedly enhanced the level of gene expression in many cell lines and the resultant MCMV promoter with the human EF-1 alpha first intron (MCMV/EF-I) was higher than the HCMV promoter from 4.3 to 65.5-fold in various cell lines. Also, the MCMV/EF-I promoter induced higher level of erythropoietin expression than the HCMV promoter in transiently transfected CHO cells.

3T3 Cells↗
PubMed2003

An optimistic view for quantifying mRNA in post-mortem human brain.

Quantitative human mRNA data are derived from post-mortem or biopsied tissue. RNA degradation, poor replication, a large mRNA variance and confounding factors such as brain pH and age of death are often cited, however, as objections to the data's reliability. A central question is whether post-mortem human mRNA can be treated as a statistically ordered system. TaqMan real-time RT-PCR was used to measure seven mRNAs in 513 cortical samples taken from 90 Alzheimer's disease and 81 control brains. Despite a high mRNA variance strong correlations were found between the mRNA transcripts in a single brain. Where a brain has a high/low level of one mRNA, the same brain invariably has a high/low level of other mRNAs; correlated order is present and allows removal of that source of variation common to all genes. Although levels of mRNA are highly variable between subjects (>1000-fold), quantitative order is present in post-mortem human mRNA, allowing effects due to pathology or gender to be isolated and tested for significance.

Aged↗
PubMed2000

Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes.

Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.

Animals↗
PubMed2001

Cooperative effects by the initiation codon and its flanking regions on translation initiation.

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Base Sequence↗
PubMed2001

Protein kinase C signalling pathway is involved in the regulation of vascular endothelial growth factor expression in human bladder transitional carcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the growth and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF(189), VEGF(165), VEGF(121)). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10(-7) M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

Carcinoma, Transitional Cell↗
PubMed1997

Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element).

Animals↗
PubMed2002

Archaeal guide RNAs function in rRNA modification in the eukaryotic nucleus.

In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years. We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.

Active Transport, Cell Nucleus↗
PubMed2002

Messenger RNA degradation: beginning at the end.

The mechanisms responsible for mRNA decay in mammalian cells, and how specific sequence elements accelerate decay, are unknown. Recent work indicates that 'ARE' instability elements recruit the exosome to promote rapid 3'-to-5' degradation of the mRNA.

3' Untranslated Regions↗
PubMed2003

Dedifferentiation of primary spermatocytes into germ cell tumors in C. elegans lacking the pumilio-like protein PUF-8.

PUF proteins are a conserved family of RNA binding proteins that regulate RNA stability and translation by binding to specific sequences in 3'-untranslated regions. Drosophila PUMILIO and C. elegans FBF are essential for self-renewal of germline stem cells, suggesting that a common function of PUF proteins may be to sustain mitotic proliferation of stem cells. Here, we show that PUF-8, the C. elegans PUF most related to PUMILIO, performs a different function in germ cells that have begun meiosis: in primary spermatocytes, puf-8 is required to maintain meiosis and prevent the return to mitosis. Primary spermatocytes lacking PUF-8 complete meiotic prophase but do not undergo normal meiotic divisions. Instead, they dedifferentiate back into mitotically cycling germ cells and form rapidly growing tumors. These findings reveal an unexpected ability for germ cells that have completed meiotic prophase to return to the mitotic cycle, and they support the view that PUF proteins regulate multiple transitions during germline development.

Animals↗
PubMed2000

A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.

Xist is required for X inactivation. To study the initiation of X inactivation, we have generated a full-length mouse Xist cDNA transgene and an inducible expression system facilitating controlled Xist expression in ES cells and differentiated cultures. In ES cells, transgenic Xist RNA was stable and caused long-range transcriptional repression in cis. Repression was reversible and dependent on continued Xist expression in ES cells and early ES cell differentiation. By 72 hr of differentiation, inactivation became irreversible and independent of Xist. Upon differentiation, autosomal transgenes did not effect counting, but transgenic Xist RNA induced late replication and histone H4 hypoacetylation. Xist had to be activated within 48 hr of differentiation to effect silencing, suggesting that reversible repression by Xist is a required initiation step that might occur during normal X inactivation in female cells.

Animals↗