Rapid mRNA Decay Research Articles

Analysis of the mechanism regulating the stability of rat macrophage inflammatory protein-2 mRNA in RBL-2H3 cells.

Using rat peritoneal neutrophils, the complete nucleotide sequence of rat macrophage inflammatory protein-2 (MIP-2) mRNA including 5'untranslated region (UTR) and 3'UTR was determined (GenBank Accession number, AB060092). It was found that the MIP-2 mRNA has a 70 bp 5'UTR, a 303 bp coding region and a 728 bp 3'UTR which contains adenylate/uridylate (AU)-rich areas defined as AU-rich elements (AREs). Site-directed mutagenesis studies using the tetracycline-sensitive transactivator protein-expressing rat basophilic leukemia cells (RBL-2H3-TO cells) revealed that MIP-2 mRNA mutants which lack the 3'UTR are more stable than MIP-2-wild-type (wt) mRNA. A MIP-2 mRNA mutant in which some mutations were introduced to the ARE was also stable. The stability of MIP-2 mRNA was low in untreated RBL-2H3-TO cells, but it increased in the antigen-stimulated immunoglobulin E (IgE)-sensitized cells. The antigen-induced MIP-2 mRNA stabilization was counteracted by the highly specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD98059. These findings indicate that ARE is the cis-element which mediates the rapid decay of MIP-2 mRNA, and the antigen stimulation stabilizes MIP-2 mRNA and the p38 MAPK and p44/42 MAPK pathways are involved in the antigen-induced stabilization of MIP-2 mRNA.

P38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

AU-rich elements (AREs) in 3'-untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated beta-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the beta-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the beta-globin reporter mRNA either lacking an ARE or containing the c-Myc 3'-untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.

A 3′ Exonuclease that Specifically Interacts with the 3′ End of Histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3′hExo, that contains a SAP and a 3′ exonuclease domain and binds the same sequence. Strikingly, 3′hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3′hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5′ side of the stem-loop region. Recombinant 3′hExo degrades RNA substrates in a 3′-5′ direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3′ end of RNA prevents its degradation by 3′hExo. These features make 3′hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

Endotoxin-adapted septic shock leukocytes selectively alter production of sIL-1RA and IL-1beta.

During septic shock, circulating levels of anti-inflammatory mediators are increased relative to those of pro-inflammatory. The reduced capacity of septic shock blood leukocytes in expressing pro-inflammatory genes in response to bacterial lipopolysaccharide endotoxin (LPS) may contribute to reductions in these mediators, but the reasons for persistent increases in circulating anti-inflammatory mediators are unknown. We determined whether septic shock leukocytes that have adapted to LPS induction of the IL-1beta gene could continue to express sIL-1RA in response to LPS. Septic shock whole-blood leukocytes and neutrophils (PMNs) selectively maintained production of sIL-1RA after treatment with LPS while limiting that of IL-1beta. Repressed transcription of IL-1beta and rapid decay of IL-1beta mRNA in septic shock neutrophils correlated with reductions in levels of IL-1beta after stimulation with LPS. Transcription of sIL-1RA mRNA was also suppressed, but the ability of LPS to stimulate events that lead to efficient translation of a stable sIL-1RA mRNA appeared responsible for maintaining sIL-1RA production. We conclude that LPS adaptation of septic shock leukocytes selectively influences signaling pathways that regulate transcription, mRNA processing, and translation, leading to changes in the balance of production of pro- and anti-inflammatory mediators.

New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

AU Binding Proteins Recruit the Exosome to Degrade ARE-Containing mRNAs

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3′ untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3′-to-5′ mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.

The Human Cytochrome P450 1A1 mRNA Is Rapidly Degraded In HepG2 Cells

The cytochromes P450 are a superfamily of enzymes that can carry out a wide range of oxidative reactions. While the transcriptional control of the cytochrome P450 genes has been relatively well-studied, posttranscriptional regulatory mechanisms that contribute to the regulation of P450s are much less well understood. We followed the decay of CYP1A1, CYP1A2, and CYP1B1 mRNAs after induction by the AH receptor ligand 2,3,7,8,-tetrachlorodibenzo-p-dioxin. CYP1A2 and CYP1B1 mRNAs were long-lived in this cell line (t12 > 24 h). In contrast, the CYP1A1 mRNA decays remarkably quickly. To determine if this rapid decay was unique to CYP1A1, we assessed the decay of selected human P450 and liver-specific mRNAs in HepG2 cells as a comparison. We analyzed albumin, phosphofructokinase, and GAPDH mRNAs and found that they were long-lived, with half-lives >24 h. We show that CYP2E1 mRNA can be detected in HepG2 cells by RT-PCR and that this mRNA also has a basal half-life of >24 h. Thus the CYP1A1 mRNA with its half-life of 2.4 h was one of the shortest-lived mRNA studied and is the most unstable of the cytochrome P450 mRNAs we have tested. The rapid decay of CYP1A1 mRNA is associated with a rapid loss in poly(A) tail length, suggesting that deadenylation is the first step in the decay pathway. The short half-life appears to be conserved across species, which suggests that this characteristic of the CYP1A1 mRNA is important for its function.

Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway.

One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult. In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery. Strategies to isolate mutants in sequence-specific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful. In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element). This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts. Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci. In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences. That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants. Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

Transcriptional and post-transcriptional regulation of monocyte chemoattractant protein-3 gene expression in human endothelial cells by phorbol ester and cAMP signalling.

Monocyte chemoattractant protein-3 (MCP-3) is one of the most broadly active chemokines, potentially inducing chemotaxis of all leucocytic cells. In the present study, we examined the regulation of MCP-3 mRNA and protein production in endothelial cells by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and cAMP signalling. On stimulation of endothelial cells with 10 nM PMA, MCP-3 mRNA increased to 300-fold the basal level at 3 hr and rapidly declined to 0.2-fold the basal level at 24 hr. PMA-induced MCP-3 mRNA and protein production of human endothelial cells were partially inhibited by pretreatment with the adenylate cyclase activator, forskolin, or membrane-permeable cAMP derivative. The PMA-induced MCP-3 mRNA increase was almost abrogated when cells were pretreated with cycloheximide (CHX). Forskolin inhibited the transcription of PMA-induced MCP-3 gene expression. Following PMA stimulation for 3 hr, subsequent addition of actinomycin D suppressed the rapid decay of PMA-induced MCP-3 mRNA. These results suggest that PMA induces the transcriptional activation of the MCP-3 gene through de novo protein synthesis and the rapid decay of PMA-induced MCP-3 mRNA through de novo synthesis of adenosine/uridine (AU)-rich element binding proteins and cAMP signalling inhibits the PMA-induced transcriptional activation of the MCP-3 gene expression.

Post-transcriptional Control of Cyclooxygenase-2 Gene Expression: THE ROLE OF THE 3′-UNTRANSLATED REGION

The cyclooxygenase (COX)-2 enzyme is responsible for increased prostaglandin formation in inflammatory states and is the major target of nonsteroidal anti-inflammatory drugs. Normally COX-2 expression is tightly regulated, however, constitutive overexpression plays a key role in colon carcinogenesis. To understand the mechanisms controlling COX-2 expression, we examined the ability of the 3'-untranslated region of the COX-2 mRNA to regulate post-transcriptional events. When fused to a reporter gene, the 3'-untranslated region mediated rapid mRNA decay (t(1/2) = 30 min), which was comparable to endogenous COX-2 mRNA turnover in serum-induced fibroblasts treated with actinomycin D or dexamethasone. Deletion analysis demonstrated that a conserved 116-nucleotide AU-rich sequence element (ARE) mediated mRNA degradation. In transiently transfected cells, this region inhibited protein synthesis approximately 3-fold. However, this inhibition did not occur through changes in mRNA stability since mRNA half-life and steady-state mRNA levels were unchanged. RNA mobility shift assays demonstrated a complex of cytoplasmic proteins that bound specifically to the ARE, and UV cross-linking studies identified proteins ranging from 90 to 35 kDa. Fractionation of the cytosol showed differential association of ARE-binding proteins to polysomes and S130 fractions. We propose that these factors influence expression at a post-transcriptional step and, if dysregulated, may increase COX-2 protein as detected in colon cancer.

A single nucleotide deletion resulting in a premature stop codon is associated with marked reduction of transcripts from a goat beta-casein null allele.

A null beta-casein allele (CSN2O) was investigated in Creole and Pyrenean goats producing milk devoid of beta-casein (CSN2). Northern blot analyses of total mammary RNA showed much lower amounts of CSN2 transcripts that were similar in size to the wild-type counterpart. The amount of CSN2O mRNA was roughly 5% of the amount of mRNA obtained at the same age and stage of lactation from CSN2A/A goats. Comparative sequence analyses of full-length CSN2O and CSN2A cDNAs showed that both alleles were of similar size, but allele CSN2O had a one-nucleotide deletion in the 5' end of exon 7, which introduces a premature stop codon. The open reading frame of allele CSN2O encodes a shortened polypeptide of 72 amino acids, compared to 223 amino acids for caprine pre beta-casein A. Comparative analyses of RT-PCR products suggested that alleles CSN2O and CSN2A might also differ in the amount and relative ratio of minor deleted CSN2 transcripts. The lower amount of CSN2O mRNA was associated with the occurrence of the premature stop codon which may mediate a rapid decay of CSN2O mRNA and promote skipping of nucleotide stretches containing premature nonsense triplets.

Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element.

AU-rich RNA-destabilizing elements (AREs) have become a paradigm for studying cytoplasmic mRNA turnover in mammalian cells. Though many RNA-binding proteins have been shown to bind to AREs in vitro, trans-acting factors that participate in the in vivo destabilization of cytoplasmic RNA by AREs remains unknown. Experiments were performed to investigate the cellular mechanisms and to identify potential trans-acting factors for ARE-directed mRNA decay. These experiments identified hnRNP D, a heterogeneous nuclear ribonucleoprotein (hnRNP) capable of shuttling between the nucleus and cytoplasm, as an RNA destabilizing protein in vivo in ARE-mediated rapid mRNA decay. Our results show that the ARE destabilizing function is dramatically impeded during hemin-induced erythroid differentiation and not in TPA-induced megakaryocytic differentiation of human erythroleukemic K562 cells. A sequestration of hnRNP D into a hemin-induced protein complex, termed hemin-regulated factor or HRF, correlates well with the loss of ARE-destabilizing function in the cytoplasm. Further experiments show that in hemin-treated cells, ectopic expression of hnRNP D restores the rapid decay directed by the ARE. The extent of destabilizing effect varies among the four isoforms of hnRNP D, with p37 and p42 displaying the most profound effect. These results demonstrate a specific cytoplasmic function for hnRNP D as an RNA-destabilizing protein in ARE-mediated decay pathway. These in vivo findings support an emerging idea that shuttling hnRNP proteins have not only a nuclear but also a cytoplasmic function in mRNA metabolism. The data further imply that shuttling hnRNP proteins define, at least in part, the nuclear history of individual mRNAs and thereby influence their cytoplasmic fate.

Effect of inflammatory cytokines on the expression of the vascular endothelial growth factor-C.

Effect of inflammatory cytokines on the expression of the vascular endothelial growth factor-C.

Cyclosporine A prevents antigen presentation by porcine endothelial cells by inhibiting TNFα-mediated class II MHC antigens expression

530 Cyclosporine A (CsA) has been so far required in the immunosuppression regimen of recipient of vascularized xenograft beyond the phase of hypercute rejection. Effects of CsA on non lymphoid immune cells such as endothelial cells (EC) are not well established and sometimes contradictory since both protective and adverse effects have been reported. In the present study, we have investigated whether CsA could contribute to alter the antigenicity of porcine aortic endothelial cells (PAEC) by reducing class I and class II MHC antigen expression. The ability of CsA to prevent MHC antigens expression during human TNFα- or lymphocytes-mediated EC activation has been evaluated by Facs and semiquantitative RT-PCR and correlated to the ability of EC to promote human lymphocyte proliferation. First, Facs analysis showed that overexpression of class II MHC antigens on PAEC, mediated by human TNFα, was strongly inhibited in the presence of CsA. The expression level of class I MHC antigens was reduced by CsA to less than 50 % of the value for EC treated with TNFα alone whereas the presence of CsA completely prevented the induction of class II MHC molecules. CsA-mediated inhibition was dose-dependent within drug concentrations ranging between 2.5 and 20 μg/ml and was consistently observed at all time points (24h to 72h) of the activation period. The presence of CsA during activation of EC with TNFα dramatically reduce the ability of EC to further promote human PBL proliferation. Indeed, when PAEC were pretreated with both TNFα and CsA, lymphocyte proliferation was below that induced by resting EC for all time points (proliferation index: 0.6, 0.2 and 0.23 for day 3, 5 and 7, respectively). Similar level of inhibition was achieved by using an anti-porcine class II MHC blocking mAb. We also observed that expression of E-selectin, as well as class I and class II porcine MHC Ag, was induced following coculture of porcine EC with human PBL. Pretreatment of porcine EC with CsA for 4h, before coculture experiments, efficiently prevented the induction of E-selectin and class II MHC antigens and inhibits overexpression of class I antigens. Semiquantitative RT-PCR experiments showed that the presence of CsA markedly reduced the steady-state level of porcine class II (SLADRA and SLADQA) mRNA at 16h as compared with PAEC stimulated with TNFα alone. This reduced level of mRNA for class II MHC was associated with the lack of class II transcriptional coactivator, CIITA, expresssion at this time point. The ability of CsA to promote the rapid decay of CIITA mRNA is analysed. Therefore, our data indicate that CsA could contribute to prevent direct presentation of porcine MHC antigens by xenogeneic EC to human T lymphocytes.

Luteinizing Hormone-Releasing Hormone (LHRH) Neurons Maintained in Hypothalamic Slice Explant Cultures Exhibit a Rapid LHRH mRNA Turnover Rate

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation.

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway. Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini-rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.

Expression and regulation of the rnc and pdxJ operons of Escherichia coli.

Escherichia coli rnc-era-recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene. RNase III, a double-stranded RNA-specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stemloop structure in the 5' untranslated leader, initiating rapid decay of the rnc operon mRNA. Here, we examine rnc operon expression and regulation in greater detail. Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP) specifies two principal mRNAs: the 1.9 kb rnc-era transcript and the less-abundant 3.7 kb RNA encoding rnc-era-recO and the downstream pdxJ and acpS genes. A 1.3 kb pdxJ-acpS RNA is transcribed from a promoter (pdxP) located within recO. About 70% of pdxJ transcription depends on transcription from rncP. Both promoters were characterized genetically. RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions. These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis-acting elements required for RNase III-initiated decay of the rnc mRNA as well as the heterologous lacZ transcript. Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site.