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PubMed1995

Third-generation model for corticosteroid pharmacodynamics: roles of glucocorticoid receptor mRNA and tyrosine aminotransferase mRNA in rat liver.

A third-generation pharmacokinetic/pharmacodynamic model was proposed for receptor/gene-mediated corticosteroid effects. The roles of the messenger RNA (mRNA) for the glucocorticoid receptor (GR) in hepatic GR down-regulation and the mRNA for hepatic tyrosine aminotransferase (TAT) induction by methylprednisolone (MPL) were examined. Male adrenalectomized Wistar rats received 50 mg/kg MPL iv. Blood and liver samples were collected at various time points for a period of 18 hr. Plasma concentrations of MPL, free hepatic cytosolic GR densities, GR mRNA, TAT mRNA, and TAT activities in liver were determined. Plasma MPL profile was biexponential with a terminal t1/2 of 0.57 hr. Free hepatic GR density rapidly disappeared from cytoplasm after the MPL dose and then slowly returned to about 60% of starting level after 16 hr. Meanwhile, GR mRNA level fell to 45% of baseline within 2 hr postdosing, and remained at that level for at least 18 hr. The GR down-regulation of GR mRNA and protein turnover rate were modeled. The TAT mRNA began to increase at about 2 hr, reached a maximum at about 5 hr, and declined to baseline by 14 hr. TAT induction followed a similar pattern, except the induction was delayed about 0.5 hr. Pharmacodynamic parameters were obtained by fitting seven differential equations in a piecewise fashion. The cascade of corticosteroid steps were modeled by a series of inductions for steroid-receptor-DNA complex, two intermediate transit compartments, TAT mRNA, and TAT activity. Results indicate that GR mRNA and TAT mRNA are major controlling factors for the receptor/gene-mediated effects of corticosteroids.

Adrenal Cortex Hormones↗
PubMed2003

Insulin down-regulates resistin mRNA through the synthesis of protein(s) that could accelerate the degradation of resistin mRNA in 3T3-L1 adipocytes.

AIMS/HYPOTHESIS: Resistin is a peptide secreted by adipocytes and recognized as a hormone that could link obesity to insulin resistance. This study was designed to examine the effect and mechanism(s) of insulin on resistin expression in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were stimulated with insulin and resistin mRNA expression was examined by Northern blot analysis. In some experiments, the insulin signal was blocked by several chemical inhibitors or overexpression of a dominant negative form (Deltap85) of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). RESULTS: Insulin treatment caused a reduction of resistin mRNA in time-dependent and dose-dependent manners in 3T3-L1 adipocytes. Pre-treatment with PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, or SB203580, an inhibitor of p38 mitogen-activated protein-kinase (p38 MAP-kinase) pathway, did not influence insulin-induced reduction of resistin mRNA. Inhibition of PI 3-kinase by LY294002 or Deltap85 also failed to block insulin-induced reduction of resistin mRNA. Cycloheximide, a protein synthesis inhibitor, completely blocked insulin-induced reduction of resistin mRNA. Actinomycin D, a RNA synthesis inhibitor, also blocked insulin-induced reduction of resistin mRNA, and the decreasing rate of resistin mRNA in cells treated with insulin alone was faster than that with actinomycin D. CONCLUSION/INTERPRETATION: Insulin downregulates resistin mRNA via PI 3-kinase, ERK or p38 MAP-kinase independent pathways in 3T3-L1 adipocytes. The downregulation mechanism of resistin mRNA by insulin would be an indirect event through the synthesis of novel protein(s) that could accelerate the degradation of resistin mRNA.

3T3 Cells↗
PubMed1991

The dynamics of the expression of C/EBP mRNA in the adult rat liver lobulus qualifies it as a pericentral mRNA.

A hybridocytochemical approach has been applied to establish whether the gene for the C/EBP mRNA might be involved in the topographical regulation of gene expression in adult rat liver. To that end the spatial distribution of the mRNA of C/EBP has been compared to that of the mRNAs of glutamine synthetase (GS), phosphoenolpyruvate carboxykinase (PEPCK) and glucokinase (GK) in normal adult livers, in livers from dexamethasone-treated animals and in livers from starved animals refed with glucose for 4 h. In normal rat liver, in situ hybridization with a probe for C/EBP mRNA revealed a low density of apparently homogeneously distributed grains, indicating low levels of C/EBP mRNA. In contrast, the livers of the experimentally-treated animals revealed a zonal distribution of the mRNA of C/EBP with the highest density of grains around the central venules. The dynamics of the pattern of expression of C/EBP mRNA are virtually identical to that of the GK mRNA. These data qualify C/EBP mRNA as a pericentral mRNA and suggest a role for the C/EBP protein in the topographical regulation of the expression of the GK mRNA.

Animals↗
PubMed1996

Opposite regulation of IGF-I and IGF-I receptor mRNA and concomitant changes of GH receptor and IGF-II/M6P receptor mRNA in human IM-9 lymphoblasts.

Human IM-9 lymphoblasts synthesize IGF-I and express IGF-I receptors, IGF-II/M6P receptors and GH receptors. We have studied the regulation of mRNA expression of IGF-I, IGF-I receptors, IGF-II/M6P receptors and GH receptors in IM-9 cells upon serum-withdrawal and re-addition of serum. IM-9 cells were cultured in RPMI-1640 medium with or without serum for various periods of time. RNA was prepared using guanidinium thiocyanate and CsCl. Antisense riboprobes for human IGF-I, IGF-I receptor, IGF-II/M6P receptor, GH receptor and for comparison for human beta-actin were synthesized and labeled with 32P. Protected fragments of 379 bases and of 420 and 350 bases with the IGF-I receptor and with the IGF-I probe respectively and protected fragments of 670 bases and of 51 and 121 bases with the GH receptor and with the beta-actin probe were detected. For the human IGF-II/M6P receptor probe protected fragments of 260 bases were visualized in RNA samples. The amount of mRNA present in each lane (10 microgram total RNA) was determined by computed densitometry. The amount of IGF-mRNA expressed by IM-9 cells decreased rapidly (within two hours) and dramatically (more than 120%) after the withdrawal of serum and increased significantly (220%) after the re-addition of serum. This increase of IGF-I mRNA preceded the increase in cell number that was seen after 48 h of medium change. Conversely, the expression of IGF-I receptor mRNA and beta-actin mRNA increased by more than 250% after the withdrawal of serum within 2 and 8 h respectively, while GH receptor mRNA fell within 2-4 h. The expression of IGF-II/M6P receptor mRNA continued to increase throughout the duration of the cell culture experiment. We conclude that IGF-I and IGF-I receptor mRNAs are regulated in an opposite direction in serum-deprived IM-9 lymphoblasts. In addition, GH receptor mRNA expression parallels IGF-I mRNA expression.

Actins↗
PubMed1996

Regulation of kappa opioid receptor mRNA in the rat brain by "binge' pattern cocaine administration and correlation with preprodynorphin mRNA.

We previously reported that 'binge' pattern administration of cocaine elevates preprodynorphin (ppDyn) mRNA in the caudate-putamen of rats. The present study confirms this finding. In addition, we report here that "binge' pattern administration of cocaine leads to a significant decrease in the mean level of kappa opioid receptor (KOR) mRNA in the substantia nigra, with no significant change in the mean level of KOR mRNA in the caudate-putamen. The decrease in KOR mRNA in the substantia nigra after 3 day or 14 day 'binge' administration of cocaine was comparable to the increase in ppDyn mRNA in the caudate-putamen. While there was no significant change in the mean levels of KOR mRNA in the caudate-putamen following cocaine administration, there was a positive within animal correlation between the levels of ppDyn mRNA and KOR mRNA in the caudate-putamen, both in animals administered saline and in animals receiving 'binge' cocaine for 14 days. Finally, mean levels of ppDyn or KOR mRNA in cocaine treated rats were not different from saline treated controls following a 10 day withdrawal from 14 days of 'binge' cocaine administration. The results provide evidence of regulation of KOR mRNA by cocaine in the substantia nigra.

Animals↗
PubMed1989

Cellular localisation of somatostatin mRNA and neuropeptide Y mRNA in foetal striatal tissue grafts.

Using in situ nucleic acid hybridisation histochemistry, we have studied the expression of somatostatin mRNA and neuropeptide Y mRNA in grafts of embryonic striatal neurones implanted into the ibotenic acid-lesioned rat neostriatum. Tissue sections of the grafted striatum were incubated with either 32P- or 35S-labelled complementary oligodeoxyribonucleotide probes specific for somatostatin mRNA and neuropeptide Y mRNA, exposed with X-ray film and dipped in Ilford K-5 emulsion. Neither somatostatin mRNA nor neuropeptide Y mRNA was detectable in the ibotenic acid-lesioned striatum indicating a pronounced degeneration of somatostatin- and neuropeptide Y-containing neurones. However, in the striatal grafts the levels of somatostatin mRNA and neuropeptide Y mRNA were substantially increased over those in the control intact striata. The results suggest that in the grafts, somatostatin mRNA and neuropeptide Y mRNA were expressed in a higher proportion of primordial striatal neurones and there was also an increased level of expression of each neuropeptide gene per individual neurone (reflecting a higher synthetic activity of such neurones) compared to the intact mature striatum. These data demonstrate the sensitivity of the in situ hybridisation technique to study patterns of gene expression in developing neuronal tissues after transplantation.

Animals↗
PubMed2004

Overall distribution of GLYT2 mRNA-containing versus GAD67 mRNA-containing neurons and colocalization of both mRNAs in midbrain, pons, and cerebellum in rats.

We aimed to clarify the overall distribution of glycinergic neurons in the midbrain, pons, and cerebellum in rats, using in situ hybridization for mRNA encoding glycine transporter 2 (GLYT2), which reliably detects glycinergic cell bodies. We combined this method with in situ hybridization for mRNA encoding glutamic acid decarboxylase isoform 67 (GAD67), and have presented for the first time global and detailed views of the distribution of glycinergic neurons in relation to GABAergic neurons. In addition to this single-detection study, we performed double-detection of GLYT2 mRNA and GAD67 mRNA to determine the distribution of neurons co-expressing these mRNAs. We have shown that many areas of the brainstem and cerebellum, not only areas where previous immunohistochemical studies have specified, involve double-labeled neurons with GLYT2 and GAD67 mRNAs. In particular, when lightly labeled GLYT2 mRNA-positive neurons were distributed within the area of GAD67 mRNA-positive neurons, almost all such GLYT2 mRNA-positive neurons were GAD67 mRNA-positive. Areas or neuron groups expressing exclusively GLYT2 mRNA or GAD67 mRNA were rather limited, such as the superior colliculus, nucleus of the trapezoid body, and Purkinje cells. The present study suggests that the corelease of glycine and GABA from single neurons is more widespread than has been reported.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Comparison between human-telomerase reverse transcriptase mRNA and alpha-fetoprotein mRNA as a predictive value for recurrence of hepatocellular carcinoma in living donor liver transplantation.

OBJECTIVE: In this study we compared the potential roles of preoperative human-telomerase reverse transcriptase (h-TERT) mRNA versus alpha-fetoprotein (AFP) mRNA expression in the peripheral blood as a tool to predict prognosis and tumor recurrence after living donor liver transplantation (LDLT) in hepatocellular carcinoma (HCC) patients. PATIENTS AND METHODS: We examined 14 patients with unresectable HCC who underwent LDLT. Six patients displayed stage IVA HCC that deviated from the Milan criteria, while the rest of the patients fell within the limitations of the Milan criteria. We analyzed the relationship between preoperative h-TERT mRNA or AFP mRNA expression in the peripheral blood and survival without recurrence. RESULTS: There was no significant difference between the survival curves without recurrence of those patients who did versus did not meet the Milan criteria. There was also no significant difference between the survival curves without recurrence among patients with positive versus negative AFP mRNA expression. However, there was a significant difference (P = .005) between the survival curves without recurrence of those patients with positive preoperative h-TERT mRNA expression versus those who either had an initially negative preoperative h-TERT mRNA or who converted from positive to negative after neoadjuvant immunochemotherapy. CONCLUSIONS: h-TERT mRNA seemed to prove more valuable than AFP mRNA not only to assess preoperative treatment modalities and postoperative patient surveillance, but also to evaluate prospective LDLT patients with HCC. Moreover, use of h-TERT mRNA could potentially expand the indications for transplantation to patients outside the Milan criteria.

Carcinoma, Hepatocellular↗
PubMed1997

Regional distribution of GABA transporter 1 (GAT1) mRNA in the rat brain: comparison with glutamic acid decarboxylase67 (GAD67) mRNA localization.

The mRNA expression of GABA transporter 1 (GAT1) was investigated in the rat brain by in situ hybridization histochemistry using an oligonucleotide probe specific to the mRNA. The pattern of GABA transporter 1 mRNA expression was well coincident with that of GABAergic terminals detected by using glutamic acid decarboxylase (GAD) as a marker. In addition, we compared the GAT1 mRNA expression with that of glutamic acid decarboxylase67 (GAD67) mRNA positive neurons. Although the expression patterns of both mRNAs were similar in most nuclei, the number of GABA transporter 1 mRNA positive cells were higher than that of GAD67 mRNA positive cells in some nuclei, such as the inferior colliculus, cerebellar nuclei and cerebral cortex. Furthermore the subthalamic nucleus and inferior olive nucleus, which lacked GAD67 mRNA expression, showed intense GABA transporter 1 mRNA expression. These data suggest that GABA transporter 1 mRNA is expressed not only in GABAergic neurons but also in non-GABAergic neurons and/or glial cells which are involved in GABAergic neurotransmission.

Animals↗
PubMed1999

TGF-beta(1), regulation of alzheimer amyloid precursor protein mRNA expression in a normal human astrocyte cell line: mRNA stabilization.

The transforming growth factor, TGF-beta(1), has been found to be increased in the central nervous system of Alzheimer's disease (AD) patients, elevates amyloid precursor protein (APP) mRNA levels in rat primary astrocytes, and may initiate or promote the deposition of amyloid-beta (Abeta) peptide in AD. Excess APP production in AD, which potentially leads to amyloidogenesis, is in part due to over expression of APP mRNA. The production of APP in a normal human cell line in contrast to transformed or animal cells provides a meaningful model to study the regulation of APP gene expression by cytokines that promotes amyloidogenesis. Here, we report that TGF-beta(1) treatment of human astrocytes markedly elevated APP mRNA levels, and also increased the half-life of APP message by at least five-fold. Under this condition, as detected by mobility shift and UV cross-linking analysis, a novel 68 kDa RNA-protein complex was formed, involving an 81 nucleotide (nt) fragment within the 3'-untranslated region (UTR), but not the 5'-UTR and coding region of APP mRNA. Insertion of the 3'-UTR onto the chloramphenicol acetyl transferase (CAT) mRNA conferred TGF-beta(1) mediated mRNA stability in transfected human astrocytes. On the other hand, the same insert carrying a deletion of the APP mRNA cis-element fragment had no effect on CAT mRNA stability. A model of APP mRNA regulation is presented in which TGF-beta(1) induced stabilization of APP message involves the binding activity of a 68 kDa RNA-protein complex within the 3'-UTR, which is likely linked to a reduction in the rate of APP mRNA decay.

3' Untranslated Regions↗
PubMed2001

Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows.

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.

Animals↗
PubMed2002

Overexpression of the 3'-untranslated region of myelin proteolipid protein mRNA leads to reduced expression of endogenous proteolipid mRNA.

The current studies focus on what mechanisms regulate the concentration of PLP mRNA in cells. The PLP mRNA is very stable and these studies suggest that its stability is regulated by a trans-acting factor specific to oligodendrocytes. In order to test whether the 3'untranslated region (3'UTR) of the PLP mRNA might regulate PLP RNA stability, C6 cells were transfected with cDNAs that expressed either luciferase or luciferase fused to the 3'UTR of PLP. Although transgene expression was low, in cells transfected with the PLP 3'UTR, there was a significant decrease in the endogenous PLP mRNA. These cells showed a distinct change in morphology and in adhesion properties. Thus, there may be a role for plp gene products in cell adhesion, which was downregulated in these cells, or an unknown function may be encoded by the PLP 3'UTR. Transgenic mice that overexpress enhanced green fluorescent protein fused to the PLP 3'UTR under control of PLP regulatory sequences were tested for the expression of the endogenous PLP mRNA. Three of four lines of transgenic mice had decreased endogenous PLP mRNA, relative to their non-transgenic littermates; the EGFP-PLP 3'UTR mouse line that expressed the highest level of transgene mRNA had a 54% reduction in PLP mRNA. We hypothesize that the PLP mRNA is regulated by elements in the 3'UTR and stabilizing proteins specific to oligodendrocytes, and that in cells that overexpress the PLP 3'UTR, these stabilizing proteins may be insufficient to maintain the normal level of the endogenous PLP mRNA.

3' Untranslated Regions↗
PubMed1992

Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain.

In situ hybridization was used to estimate regional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was increased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distribution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (1-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide-spread increase in HSP-70 mRNA suggests that reperfusion triggered expression in all previously ischemic regions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the ischemic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions undergoing injury, but is transient in surrounding regions that recover.

Animals↗
PubMed1977

Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA.

We have compared the behavior of a prokaryotic mRNA in a eukaryotic ribosome binding system and of a eukaryotic mRNA in a prokaryotic ribosome binding system. Using (32)P- and (125)I-labeled bacteriophage f1 mRNA, we have shown that rabbit reticulocyte 80S ribosomes can protect specific sequences from pancreatic RNase digestion, including those sequences protected by Escherichia coli ribosomes. We have also found that E. coli ribosomes fail to protect any region of (125)I-labeled globin mRNA. Iodination of the mRNA appeared to have little or no effect on the specificity of binding or protection by the ribosomes of either system.The eukaryotic and prokaryotic systems differ markedly in the ability of the small ribosomal subunits to protect mRNA from nuclease digestion. The regions of phage f1 mRNA protected by E. coli 30S subunits are virtually identical to those protected by the 70S ribosomes. By contrast, rabbit reticulocyte 40S subunits protect substantially larger fragments of mRNA from nuclease digestion than do the 80S ribosomes. These 40S-protected fragments are specific in the case of globin mRNA and overlap the shorter region protected by the 80S ribosomes. However, the 40S-protected fragments of phage f1 mRNA were found to be extremely heterogeneous, reflecting perhaps an important difference between the initial interactions made by these two mRNAs with the ribosomes.

Animals↗
PubMed2002

Post-transcriptional regulation of luteinizing hormone receptor mRNA in the ovary by a novel mRNA-binding protein.

Luteinizing hormone (LH) receptor mRNA is post-transcriptionally regulated. An ovarian cytosolic LH receptor mRNA-binding protein (LRBP) identified in our laboratory binds to a polypyrimidine-rich bipartite sequence in the coding region of LH receptor mRNA. The present studies show a role for LRBP in the regulation of LH receptor mRNA. We demonstrated that increased LH receptor mRNA degradation occurs during hormone-induced LH receptor down-regulation. Furthermore, increased degradation of LH receptor mRNA was seen when partially purified LRBP was included in an in vitro mRNA decay reaction. The LH receptor mRNA binding activity of LRBP measured by RNA electrophoretic mobility shift analysis showed an inverse relationship to LH receptor mRNA levels during different physiological states. These results suggest that LRBP is a physiological regulator of LHR mRNA expression in the ovary and provides a novel mechanism for the regulation of LH receptor expression in the ovary.

Animals↗
PubMed2004

Selective cytoplasmic translocation of HuR and site-specific binding to the interleukin-2 mRNA are not sufficient for CD28-mediated stabilization of the mRNA.

The interleukin-2 mRNA is a labile transcript containing AU-rich elements that is transiently stabilized by CD28 receptor signaling. For a number of proto-oncogenes and cytokines, the HuR protein has been shown to avidly bind the AU-rich elements that confer instability upon their mRNAs. HuR was originally thought to participate in mRNA degradation but subsequent studies indicated that it actually functions to stabilize mRNA. Binding of HuR to the mouse interleukin-2 mRNA has not been studied. We tested if HuR binds the interleukin-2 mRNA and whether such binding is related to CD28-mediated stabilization of the mRNA. First, we confirm that T cell receptor signaling, which is sufficient to induce interleukin-2 transcription, also triggers translocation of HuR from the nucleus to the cytoplasm. Interestingly, T cell receptor-triggered translocation is selective as heterogeneous nuclear ribonucleoprotein A1 does not shuttle under the same conditions. Engagement of both the T cell and CD28 receptors, which enhance interleukin-2 transcription and induce stabilization of the mRNA, did not further increase the level of cytoplasmic HuR. Using an in vitro binding assay, we demonstrate that HuR binds the interleukin-2 mRNA and localize binding to a sequence downstream of the single nonameric AU-rich element that is present in its 3'-untranslated region. However, we conclude that HuR binding to the interleukin-2 mRNA, both in vitro and in vivo, is not associated with alterations in mRNA stability.

Animals↗
PubMed2002

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.

Actins↗
PubMed1995

mRNA decay in spinach chloroplasts: psbA mRNA degradation is initiated by endonucleolytic cleavages within the coding region.

The expression of chloroplast genes during leaf development in higher plants is regulated on several levels as transcription, RNA processing and stability, protein stability and turnover. Differential mRNA stability is one major component which contributes to the developmentally controlled accumulation of higher plant chloroplast psbA mRNA, which encodes the D1 protein of photosystem II. To understand the molecular mechanisms of specific mRNA degradation an in vitro mRNA decay system based on lysed chloroplasts from spinach leaves was established. Employing this degradation extract the decay of psbA mRNA was analyzed. Half-life of the psbA mRNA in vitro is dependent on the degradation conditions as the presence of Mg2+, which was found to stabilize the mRNA. Addition of tRNA stabilizes the mRNA and allows the accumulation of distinct degradation intermediates. psbA mRNA derived fragments of the same size were detected in degradation experiments in vitro, in organello and in vivo. 5' ends of the degradation intermediates were identified by primer extension and found to be localized in the 5' part of the coding region. The data indicate a degradation mechanism involving initiation of psbA mRNA decay by specific endonucleolytic cleavage and subsequent exonucleolytic degradation of the fragments. Possible models for cleavage site recognition are discussed.

Base Sequence↗