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PubMed2001

Arc mRNA dynamics: return to sender--the NMDA receptor provides the targeting address for Arc mRNA.

The recently defined role for local protein synthesis during long-lasting synaptic plasticity has focused attention on the mechanisms underlying dendritic mRNA localization. The mRNA encoded by the immediate-early gene Arc is not only transported into dendrites, but localizes specifically to activated postsynaptic sites. This specific localization has now been shown to depend on local signaling through the NMDA receptor.

Animals↗
PubMed2003

Distribution of glycine transporter 2 mRNA-containing neurons in relation to glutamic acid decarboxylase mRNA-containing neurons in rat medulla.

We studied the distribution of medullary glycinergic neurons in relation to GABAergic neurons, by using in situ hybridization method for mRNA encoding either glycine transporter 2 (GLYT2) or glutamic acid decarboxylase isoform 67 (GAD67). GLYT2 mRNA-positive (GLYT2+) neurons were distributed widely and clustered in (1). the respiration-related area of the ventrolateral medulla called the Bötzinger complex, (2). the nucleus retroambiguus caudal to the obex or the caudal ventral respiratory group, (3). the spinal trigeminal nucleus, (4). a small area immediately dorsal to the inferior olivary nucleus, and (5). the border zone between the hypoglossal nucleus and the surrounding reticular formation. It was characteristic that in the dorsomedial medulla, GLYT2+ neurons were distributed only sparsely in contrast to dense GAD67+ neurons. Only few GLYT2+ neurons were distributed in the medial and interstitial subnuclei of the nucleus tractus solitarii. In particular virtually no GLYT2+ neurons were found in the area postrema. Furthermore, in the reticular formation and the spinal trigeminal nucleus, GAG67+ neurons tended to be distributed in the area where GLYT2+ neurons were sparse, and vice versa. These results provide useful information for the effort of determining neurotransmitters involved in the medullary neurons.

Amino Acid Transport Systems, Neutral↗
PubMed1997

Growth hormone down-regulates growth hormone receptor mRNA in chickens but developmental increases in growth hormone receptor mRNA occur independently of growth hormone action.

The purpose of this study was to determine the role of growth hormone (GH) in regulating expression of the chicken GH receptor (cGHR) gene by comparing the levels of cGHR mRNA in livers of normal chickens with that of GHR-deficient dwarf chickens. Since the sex-linked dwarf chicken lacks a functional cGHR, there are no genes activated as a result of GH action. Examination of the early developmental profile of hepatic cGHR mRNA in normal and dwarf chickens should yield information on the relative contribution of developmental and hormonal factors to the regulation of cGHR gene expression. Using a sensitive RNase protection assay, we found that the abundance of the major cGHR transcripts (4.3, 3.2 and 0.8 kb) in normal chickens increases about 2-fold between 1 and 7 weeks of age. Due to a splice site mutation in the dwarf chicken, the two larger transcripts encoding the full-length cGHR are not expressed. However, the expression of the truncated cGHR transcript (0.8 kb) in dwarf chickens increases about 5-fold between 1 and 7 weeks of age which suggests that the cGHR gene is overexpressed when not down-regulated by GH. Furthermore, a single promoter, appears to control expression of cGHR transcripts in liver since primer extension analysis revealed the same 5'-end in both full-length and 0.8 kb transcripts. These observations suggest that even though developmental increases in cGHR gene expression occur independently of GH action, GH, either directly or indirectly, down-regulates expression of the cGHR gene in normal chickens.

Animals↗
PubMed2001

Prepro-orexin mRNA levels in the rat hypothalamus, and orexin receptors mRNA levels in the rat hypothalamus and adrenal gland are not influenced by the thyroid status.

Orexins are two recently discovered neuropeptides that play an important role in the regulation of food intake and in the regulation of the sleep-wake cycle. In this work we examined the effects of thyroid hormones on prepro-OX expression in the rat hypothalamus, and OXRs expression in the rat hypothalamus and adrenal gland. Hypo- and hyperthyroidism were induced in adult male rats, and the levels of hypothalamic prepro-OX and OXRs mRNA, and adrenal OXRs mRNA were determined using semiquantitative reverse transcription-polymerase chain reaction and/or in situ hybridization. Our results indicate that thyroid status affects neither prepro-OX in the hypothalamus nor hypothalamic and adrenal gland OXRs expression.

Adrenal Glands↗
PubMed1997

Downregulation of corticotropin-releasing factor mRNA, but not vasopressin mRNA, in the paraventricular hypothalamic nucleus of rats following nutritional stress.

Stress can cause disturbance of homeostasis to result in illness. Stress can also induce various gene expression in different neuronal systems. For example, nutritional stress induced by acute food deprivation upregulates corticotropin-releasing factor (CRF) mRNA, whereas osmotic stress increases vasopressin (VP) mRNA. However, it is unknown if nutritional stress induced by chronic food deprivation has synergistic effects on CRF and VP mRNAs. We have used in situ hybridization in conjunction with quantitative autoradiography to demonstrate that nutritional stress induced by a 4-day food deprivation results in a body-weight loss with a significant decrease of CRF mRNAs, but not VP mRNAs in the paraventricular hypothalamic nucleus (PVN) of Sprague-Dawley rats. The present study has thus indicated that a chronic nutritional stress does not have synergistic effects on CRF and VP mRNAs. The decrease of CRF mRNAs is obviously related to the body-weight loss induced by food deprivation. This study thus supports a notion that the CRF, but not VP, neurons in the PVN play an important role in their neuroadaptation associated with body weight loss. Thus, it is conceivable that downregulated CRF neurons in the hypothalamus could be involved in pathogenesis of human eating disorder with severe weight loss, whereas upregulated CRF neurons could be associated with an opposite form of the eating disorder that causes obesity.

Animals↗
PubMed1991

Butyrate stimulates the secretion of apolipoprotein (apo) A-I and apo B100 by the human hepatoma cell line Hep G2. Induction of apo A-I mRNA with no change of apo B100 mRNA.

Addition of sodium butyrate to the culture medium of the human hepatoma cell line Hep G2 resulted in a time- and dose-dependent increase in the secretion of apolipoprotein A-I (apo A-I) and apolipoprotein B100 (apo B100). After a 24 h preincubation period, a 2.4- and 2.2-fold increase in the secretion of apo A-I and apo B100 respectively was obtained during the next 24 h in the presence of 2 mM-sodium butyrate. Secretion of albumin, fibrinogen or [35S]methionine-labelled newly synthesized proteins was unaffected or only marginally affected, indicating that the effect of butyrate on apo A-I and apo B100 is not part of a general effect on protein synthesis and secretion. In structure-function studies, butyrate was found to be the most potent inducer among various straight-chain carboxylic acids. Hydroxylated, aminated and otherwise modified butyrate derivatives were inactive. The enhanced accumulation of apo A-I and apo B100 in the culture medium could not be explained by changes in the uptake and degradation of the synthesized apolipoproteins or by alterations in the secretion of possible intracellular pools. In addition, [35S]methionine incorporation studies indicated that synthesis and/or secretion of newly synthesized apo A-I and apo B100 is enhanced in the presence of butyrate. The apo A-I mRNA level was increased 2.3-fold upon treatment with 2 mM-butyrate for 48 h, suggesting regulation at (post-)transcriptional level. In contrast, no change in the level of apo B100 mRNA in butyrate-treated cells was observed, indicating regulation at translational or co- or post-translational level. We propose that the effect of butyrate on the secretion of apo A-I and apo B100 by Hep G2 results from two different regulatory mechanisms.

Albumins↗
PubMed2001

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum.

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.

Amino Acid Transport System X-AG↗
PubMed1999

sst1 mRNA is the prominent somatostatin receptor mRNA in the rat gastrointestinal tract: reverse transcription polymerase chain reaction and in situ-hybridization study.

The inhibitory peptide hormone somatostatin and its receptors (sst1-sst5) regulate many physiological functions in the gastrointestinal tract. In an attempt to correlate the various effects of somatostatin in gastrointestinal physiology to individual sst subtypes sst1-sst5, mRNAs have been localized by semiquantitative reverse transcription polymerase chain reaction amplification and in situ hybridization of sst1 and sst3 in the rat alimentary tract. sst1-sst4 mRNAs were found throughout the gastrointestinal tract, sst1 mRNA being more abundant than sst2 and much more abundant than sst3 and sst4 mRNAs. sst5 transcripts were at the detection threshold. sst1 and sst3 mRNAs are present in enterocytes and enteric neurons suggesting a role of these subtypes in the somatostatin-mediated inhibition of acetylcholine release from myenteric neurons and of secretomotor neuron activity in the submucous plexus. The presence of sst3 mRNA in smooth muscle cells points to an additional role of this receptor in regulating gut motility.

Animals↗
PubMed1990

Testosterone increases TSH-beta mRNA, and modulates alpha-subunit mRNA differentially in mouse thyrotropic tumor and castrate rat pituitary.

TSH, LH and FSH, the three pituitary glycoprotein hormones, are each composed of a common alpha-subunit and a hormone specific beta-subunit. Testosterone is known to regulate all three intact hormones differently in the rodent. However, there is only one gene encoding the common alpha-subunit. In order to elucidate the effects of testosterone on TSH subunit synthesis and its regulation of the common alpha-subunit, two in vivo models were studied: castrate rat pituitary was used as a gonadotropin-enriched tissue; and mouse thyrotropic tumor was used as a thyrotropin-enriched tissue. Male castrate rats were treated with testosterone propionate, 500 micrograms/100 g BW, sc, for 11 days. Testosterone increased plasma TSH to 131% of control values (P less than 0.02), while plasma LH fell to undetectable levels, and plasma alpha-subunit fell to 14% of control values (P less than 0.001). Testosterone increased TSH-beta mRNA to 237% of control values (P less than 0.02), while alpha-subunit mRNA fell to 20% of control values (P less than 0.001). Hypothyroid mice bearing thyrotropic tumors were treated with testosterone propionate, 150 micrograms/100 g BW, sc, for 11 days. In this model plasma TSH-beta and alpha-subunit concentrations are 1000-fold higher than in non-tumor bearing animals, and the contribution of pituitary gonadotropes to plasma subunit concentrations is negligible. "Total" TSH-beta and alpha-subunit concentrations were estimated as one-half of intact TSH plus the respective free subunit concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli.

Initiation complexes formed by E. coli ribosomes in the presence of 32P-labeled A protein initiator region from R17 bacteriophage Rna have been treated with colicin E3 and disassembled by exposure to 1% sodium dodecyl sulfate. Electrophoresis on 9% polyacrylamide gels reveals a dissociable complex containing the 30-nucleotide-long messenger fragment and the 50-nucleotide-long colicin fragment, which arises from the 3' terminus of the 16S RNA. The complex is a pure RNA-RNA hybird; it is apparently maintained by a seven-base complementarity between the two RNA fragments. Detection of this mRNA-rRNA complex strongly supports the hypothesis that during the initiation step of protein biosynthesis the 3' end of 16S RNA base pairs with the polypurine stretch common to initiator regions in E. coli and bacteriophage mRNAs. The implications of our findings with respect to the molecular mechanism of initiation site selection and mRNA binding to ribosomes, the role of rRNA in ribosome function, and species specificity in translation are explored.

Base Sequence↗
PubMed1980

Vero cells injected with adenovirus type 2 mRNA produce authentic viral polypeptide patterns: early mRNA promotes growth of adenovirus-associated virus.

Adenovirus type 2 mRNAs were injected via glass capillaries into Vero cells, a line of African green monkey kidney cells permissive for adenovirus growth. Polypeptides synthesized after injection were labeled with 35S-labeled amino acids, precipitated with antiviral sera, and analyzed by polyacrylamide gel electrophoresis. Both early and late viral mRNAs give rise to authentic polypeptides. The in vivo translation of mRNAs can be measured as late as 24 hr after injection. The ability to analyze the protein products of microinjected mRNAs directly should greatly extend the applications of the procedure. Vero cells injected with early mRNA from adenovirus type 2 support the growth of adenovirus-associated virus, a defective virus that is dependent on adenovirus helper functions. This result demonstrates that a measurable biological activity, the ability to overcome the defectiveness of adenovirus-associated virus, resides in early adenovirus mRNA.

Adenoviruses, Human↗
PubMed1983

Role of mRNA competition in regulating translation: further characterization of mRNA discriminatory initiation factors.

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected of the mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a "cap binding protein" complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor.

Animals↗
PubMed1984

Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.

Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.

Adenoviruses, Human↗
PubMed1990

Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast.

In yeast, the mRNA encoded by the MAT alpha 1 gene is unstable (t1/2 = 5 min) and the mRNAs encoded by the ACT1 gene (t1/2 = 30 min) and the PGK1 gene (t1/2 = 45 min) are stable. To understand the RNA structural features that dictate mRNA decay rates in yeast, we have constructed PGK1/MAT alpha 1 and ACT1/MAT alpha 1 gene fusions and analyzed the decay rates of the resultant chimeric transcripts. Fusion of a MAT alpha 1 segment containing 73% of the coding region and the 3' untranslated region to either of the stable genes is sufficient to cause rapid decay of the chimeric mRNAs (t1/2 = 6-7.5 min). Sequences required for this rapid decay are not found in the MAT alpha 1 3' untranslated region but are located within a 42-nucleotide segment of the coding region that has a high content (8 out of 14) of rare codons. Introduction of a translational stop codon upstream of this region stabilizes the hybrid mRNAs, indicating that the rapid decay promoted by these sequences is dependent on ribosomal translocation.

Base Sequence↗
PubMed1999

Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae.

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

5' Untranslated Regions↗
PubMed1989

Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site.

Three different mRNA analogues (28 to 34 nucleotides long) were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 5'-side of these coding triplets. A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide. The messages were bound to E. coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated. Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA. Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked. The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus. These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction.

Base Sequence↗
PubMed1991

Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs.

Amino Acid Sequence↗
PubMed1999

A conditional U5 snRNA mutation affecting pre-mRNA splicing and nuclear pre-mRNA retention identifies SSD1/SRK1 as a general splicing mutant suppressor.

A combination of point mutations disrupting both stem 1 and stem 2 of U5 snRNA (U5AI) was found to confer a thermosensitive phenotype in vivo. In a strain expressing U5AI, pre-mRNA splicing was blocked before the first step through an inability of the mutant U5 snRNA to efficiently associate with the U4/U6 di-snRNP. Formation of early splicing complexes was not affected in extracts prepared from U5 snRNA mutant cells, while the capacity of these extracts to splice a pre-mRNA in vitro was greatly diminished. In addition, significant levels of a translation product derived from intron containing pre-mRNAs could be detected in vivo. The SSD1/SRK1 gene was identified as a multi-copy suppressor of the U5AI snRNA mutant. Single copy expression of SSD1/SRK1 was sufficient to suppress the thermosensitive phenotype, and high copy expression partially suppressed the splicing and U4/U6.U5 tri-snRNP assembly pheno-types. SSD1/SRK1 also suppressed thermosensitive mutations in the Prp18p and U1-70K proteins, while inhibiting growth of the cold sensitive U1-4U snRNA mutant at 30 degrees C. Thus we have identified SSD1/SRK1 as a general suppressor of splicing mutants.

Base Sequence↗