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PubMed1991

A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability.

A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed.

Base Sequence↗
PubMed2002

Increasing maize seed methionine by mRNA stability.

The amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein-bound methionine. This variability is a result of post-transcriptional regulation of the Dzs10 gene, which encodes a seed-specific high-methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis-acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.

Amino Acids↗
PubMed2000

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Animals↗
PubMed1991

An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro.

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

Adenine↗
PubMed1989

Control of mRNA stability by the virion host shutoff function of herpes simplex virus.

vhs1 is a mutant of herpes simplex virus type 1 that is defective in the virion host shutoff function responsible for the degradation of cellular mRNAs and the concomitant shutoff of host protein synthesis. In this study, the effect of the vhs1 mutation on the metabolism of viral mRNAs was examined by measuring the half-lives and patterns of accumulation of 10 different viral mRNAs representing all kinetic classes. The vhs1 mutation had the effect of dramatically lengthening the cytoplasmic half-lives of all 10 mRNAs. In wild-type virus infections, the 10 mRNAs had similar half-lives, suggesting that little, if any, target mRNA selectivity was exhibited by the vhs function. The vhs1 mutation caused overaccumulation of a number of mRNAs. The effect was most dramatic for the alpha (immediate-early) mRNA for ICP27 and the beta (early) mRNAs encoding thymidine kinase, ICP8, and DNA polymerase. Whereas in wild-type infections these mRNAs increased to peak levels and subsequently declined in abundance, in vhs1 infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and gamma (late) mRNAs. The results suggest that the vhs protein plays an important role in determining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal downregulation at late times of alpha and beta gene expression.

Animals↗
PubMed2003

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels.

The pnp gene, encoding the enzyme polynucleotide phosphorylase (PNPase), was overexpressed in the actinomycin producer Streptomyces antibioticus. Integration of pIJ8600, bearing the thiostrepton-inducible tipA promoter, and its derivatives containing pnp into the S. antibioticus chromosome dramatically increased the growth rate of the resulting strains as compared with the parent strain. Thiostrepton induction of a strain containing pJSE340, bearing pnp with a 5'-flanking region containing an endogenous promoter, led to a 2.5-3 fold increase in PNPase activity levels, compared with controls. Induction of a strain containing pJSE343, with only the pnp ORF and some 3'-flanking sequence, led to lower levels of PNPase activity and a different pattern of pnp expression compared with pJSE340. Induction of pnp from pJSE340 resulted in a decrease in the chemical half-life of bulk mRNA and a decrease in poly(A) tail length as compared to RNAs from controls. Actinomycin production decreased in strains overexpressing pnp as compared with controls but it was not possible to attribute this decrease specifically to the increase in PNPase levels. Overexpression of pnp had no effect on ppGpp levels in the relevant strains. It was observed that the 3'-tails associated with RNAs from S. antibioticus are heteropolymeric. The authors argue that those tails are synthesized by PNPase rather than by a poly(A) polymerase similar to that found in Escherichia coli and that PNPase may be the sole RNA 3'-polynucleotide polymerase in streptomycetes.

Anti-Bacterial Agents↗
PubMed1996

A common frameshift mutation in von Willebrand factor does not alter mRNA stability but interferes with normal propeptide processing.

Quantitative defects in von Willebrand factor (VWF) result in type 1 and type 3 von Willebrand disease (VWD). This study characterizes the defect in VWF expression resulting from a single nucleotide deletion in VWF exon 18, a mutation previously reported to be common among type 3 VWD patients. A severely affected (type 3) VWD patient in the current pedigree is homozygous for the mutation, whereas heterozygous individuals exhibit variable expression of type 1 VWD. In contrast to the previously reported high frequency of the exon 18 deletion in Sweden and Germany, this mutation appears to be infrequent among type 3 VWD patients in the United States. Although this frameshift mutation results in proximal premature termination of VWF translation, the abnormal VWF mRNA is stable. The mutant truncated recombinant VWF protein is retained within the transfected cell, and no propeptide processing is observed, suggesting a defect in protein folding. Cotransfection of mutant and wild-type recombinant VWF fails to demonstrate a dominant effect of the mutant on the normal allele. Consistent with these results, plasma VWF propeptide of the homozygous individual was markedly reduced whereas heterozygotes exhibited moderately reduced levels. In contrast to type 2A VWD (group 1), the misfolded mutant protein does not appear to exert a dominant-negative effect on normal VWF subunits expressed from the wild-type allele.

Exons↗
PubMed1999

Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein L.

A 126-base region of human vascular endothelial growth factor (VEGF) 3'-untranslated region, which we identified as the hypoxia stability region, forms seven hypoxia-inducible RNA-protein complexes with apparent molecular masses ranging from 40 to 90 kDa in RNA-UV-cross-linking assays. In this study, we show that proteins that form the 60-kDa RNA-protein complex with the hypoxia stability region were present in both cytoplasmic and nuclear compartments. We purified the protein associated in the 60-kDa complex and identified it as heterogeneous nuclear ribonucleoprotein L (hnRNP L) by protein sequencing. Removal of hnRNP L by immunoprecipitation specifically abolished formation of the 60-kDa complex. Synthetic deoxyribonucleotide competition studies defined the RNA-binding site of hnRNP L as a 21-base-long sequence, 5'-CACCCACCCACAUACAUACAU-3'. Immunoprecipitation of hnRNP L followed by reverse transcription-polymerase chain reaction showed that hnRNP L specifically interacts with VEGF mRNA in hypoxic cells in vivo. Furthermore, when M21 cells transfected with antisense oligodeoxyribonucleotide to the hnRNP L RNA-binding site, the VEGF mRNA half-life was significantly reduced under hypoxic conditions. Thus, we propose that specific association of hnRNP L with VEGF mRNA under hypoxia may play an important role in hypoxia-induced post-transcriptional regulation of VEGF mRNA expression.

Base Sequence↗
PubMed2001

Decreased mRNA stability as a mechanism of glucocorticoid-mediated inhibition of vascular endothelial growth factor gene expression by cultured keratinocytes.

Epidermal keratinocyte-derived overexpression of vascular endothelial growth factor has been functionally linked to increased density of tortuous and hyperpermeable dermal microvessels, representing a characteristic component of cutaneous inflammation. We hypothesized that potent anti-inflammatory properties of synthetic glucocorticoids are attributed in part to their interference with the regulated vascular endothelial growth factor expression by keratinocytes. As vascular endothelial growth factor is markedly upregulated by autocrine transforming growth factor alpha and paracrine hepatocyte growth factor/scatter factor expression, the effect of glucocorticoids on growth-factor-induced vascular endothelial growth factor production by primary and immortalized keratinocytes was examined. Glucocorticoids were shown to suppress vascular endothelial growth factor protein and mRNA expression in a concentration- and time-dependent fashion. In transcriptional activation studies, however, common 5'-regulatory regions of the vascular endothelial growth factor gene failed to confer inhibitory glucocorticoid effects. Instead, glucocorticoids were shown to increase vascular endothelial growth factor mRNA turnover, indicating that post-transcriptional modes of glucocorticoid action are employed to negatively regulate induced vascular endothelial growth factor expression. Together, these studies identify vascular endothelial growth factor upregulation by epidermal keratinocytes as a putative target of glucocorticoid action in cutaneous inflammation. Our data provide strong evidence that mRNA destabilization may represent a mechanism by which glucocorticoids inhibit growth-factor-regulated vascular endothelial growth factor gene expression by keratinocytes.

Adult↗
PubMed1989

Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation.

Tubulin synthesis in animal cells is controlled in part by an autoregulatory mechanism that modulates the stability of ribosome-bound tubulin mRNAs. For beta tubulin, the initial recognition event for this selective RNA instability has previously been shown to be a cotranslational binding (presumably by tubulin itself) to the nascent amino-terminal beta-tubulin tetrapeptide just after it emerges from the ribosome. Although this "autoregulation" of tubulin expression is thus obligatorily linked to the translation process, the mechanism of how a cotranslational protein-protein binding event ultimately triggers RNA degradation is unknown. Using protein synthesis inhibitors to slow and ultimately to block translation elongation, we now show that the mRNA destabilization pathway requires ongoing ribosome translocation.

Animals↗
PubMed2005

Reduced stress tolerance of glutamine-deprived human monocytic cells is associated with selective down-regulation of Hsp70 by decreased mRNA stability.

In critically ill patients, clinicians observe a reverse correlation of survival and a decreased plasma concentration of the most abundant free amino acid, glutamine (Gln). However, in this context, the role of Gln remains largely elusive. Gln is used as an energy substrate by monocytes. Gln deprivation of these cells results in an increased susceptibility to cell stress and apoptosis, as well as in a reduced responsiveness to pro-inflammatory stimuli. We performed a systematic study to elucidate the molecular mechanism by which Gln depletion affects the heat stress response of the monocytic cell line U937. Proteomic analysis revealed that Gln depletion was associated with specific changes in the protein expression pattern. However, the overall level of tRNA-bound Gln remained unaffected. The stress protein heat shock protein (Hsp) 70 showed the highest reduction in protein synthesis. This was due to enhanced mRNA decay during Gln starvation while the transcriptional and the translational control of Hsp70 expression remained unchanged. A physiological Gln concentration and above was found to be necessary for maximum Hsp70 accumulation upon heat shock. Thus, the study shows a specific link between Gln metabolism and the regulation of heat shock proteins.

Cells, Cultured↗
PubMed1999

Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization.

Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the beta-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11-389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.

Bronchi↗
PubMed1999

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.

3' Untranslated Regions↗
PubMed1999

Sustained elevated levels of VCAM-1 in cultured fibroblast-like synoviocytes can be achieved by TNF-alpha in combination with either IL-4 or IL-13 through increased mRNA stability.

Rheumatoid arthritis is characterized by hyperplasia of the synovial lining and invasion of cartilage and bone by a subset of resident synovial cells named fibroblast-like synoviocytes. They are characterized by elevated expression of the vascular cell adhesion molecule-1 (VCAM-1). The intensity of VCAM-1 expression correlates with the degree of inflammation of the synovial joint. Differential VCAM-1 expression may determine inflammatory cell accumulation through its interaction with leukocytes that express the counterreceptor integrins alpha4beta1 and alpha4beta7. Elevated levels of VCAM-1 expression are thought to be a consequence of the presence of inflammatory mediators, in particular IL-1beta and TNF-alpha. Fibroblast-like synoviocytes rapidly up-regulate VCAM-1 expression in response to IL-1beta and TNF-alpha, but also to IL-4. However, we now show that the response to IL-1beta or TNF-alpha is of a brief transient nature, even when applied continuously over a period of 12 days, whereas the response to IL-4 or IL-13 is sustained. Great synergy is obtained by combining either IL-4 or IL-13 with TNF-alpha, which results in a highly elevated but also sustained expression of VCAM-1. The mechanism by which IL-4 or IL-13 prolongs VCAM-1 expression can be explained by a dramatic increase in the half-life of VCAM-1 mRNA.

Arthritis, Rheumatoid↗
PubMed1996

Analysis of the rat clusterin gene promoter and cyclic AMP-regulated mRNA stability in testicular cells.

Clusterin, also known as SGP-2 or TRPM-2, is expressed in the male reproductive tissues at different levels. The genomic structure of the rat clusterin gene was recently reported by our laboratory and others. In this study, we have determined the promoter responsible for the basal expression of the rat clusterin gene in testicular cells by analyzing the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in MA-10 cells driven by different segments of the 5'-flanking region and the first intron of the clusterin gene. The region required for maximal basal expression was identified at -266 to +54. Addition of DNA fragments of the rat clusterin gene from -1298 to -266 bp, or from +54 to +1153 to (-266/+54)CAT resulted in a 87% decrease in CAT activity, suggesting the presence of inhibitory DNA elements in both the 5'-flanking region and the first intron. When DNA fragment in the first intron, +1153 to +2874, was included, CAT activity in the (-266/+2874)CAT construct increased to 70% of the clusterin promoter (-266/+54)CAT, indicating that stimulatory DNA elements may be present in this region of the first intron. Treatment of MA-10 cells with cyclic AMP (cAMP) neither decreased CAT activity driven by any of the clusterin/CAT chimeric plasmids examined in transient transfection studies, nor reduced the synthesis of nuclear clusterin RNA in nuclear run-on assays, indicating that the reduction of clusterin mRNA levels by cAMP previously reported in our laboratory is not exerted at the transcriptional level. Furthermore, addition of transcriptional or translational inhibitors (actinomycin D and cycloheximide respectively) abolished the cAMP effect observed in MA-10 cells. In summary, we have demonstrated that the basal transcription of the rat clusterin gene in testicular cells is under the control of both positive and negative regulatory sequences at the 5'-flanking region as well as in the first intron. The reduction of clusterin mRNA after exposure of MA-10 cells to cAMP is not due to a decrease in its transcriptional activity, but rather to an increase in the degradation of this mRNA through synthesis of a destabilizing protein(s) and its mRNA.

Animals↗
PubMed2002

Analysis of involvement of the 3'-untranslated regions in regulating mRNA stability for vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus.

The degradation of the 3'-untranslated regions (UTRs) of vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus, was analyzed in vitro. The degradation pattern was similar for all three RNAs, with a high degradation rate in non-diapausing adult insects and no degradation in the fifth instar nymphs and in diapausing adults, and was not correlated with the expression levels of these three proteins. Proteins binding to the 3'-UTRs were detected in polysomal and cytosolic extracts. These factors, however, were present in all developmental stages. The abundance of the polysomal factor showed little variation, but the cytosolic factor was enriched in adult insects. Cross-competition experiments demonstrated that the same factors bound to all three RNAs with similar affinity. The pattern of degradation, presence of the binding factors in all stages, and their inability to distinguish between the target sequences indicate that the 3'-UTRs do not participate in controlling the expression of these three proteins.

3' Untranslated Regions↗
PubMed2004

How adhesion, migration, and cytoplasmic calcium transients influence interleukin-1beta mRNA stabilization in human monocytes.

We investigated the mechanisms by which primary human monocyte migration and the production of important cytokines are co-regulated. Motile monocytes underwent cyclic morphologic and adhesive changes that were associated with intracellular free calcium changes; in such cells, cytokine transcripts were unstable and translationally repressed. Agents that activate monocytes, including lipopolysacharrides (LPS), cytomegalovirus (CMV), and tumor necrosis factor (TNFalpha), have been shown to de-repress translation and these agents stabilize adhesion-induced transcripts for IL-lbeta and IL-8 and markedly diminish cell migration in the presence of autologous serum. LPS suppressed Rho A activity and either this agent or C3 transferase elevated intracellular free calcium, stabilized transcripts, and, in tandem, inhibited cell migration by preventing tail retraction, a prerequisite for cell translocation. These results, therefore, suggest that monocyte activating agents inhibit the RhoA pathway and continuously elevate intracellular calcium leading to a concomitant decrease in monocyte migration and stabilization of cytokine transcripts prior to translation.

ADP Ribose Transferases↗