Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mRNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed2000

Length increase of the human alpha -globin 3'-untranslated region disrupts stability of the pre-mRNA but not that of the mature mRNA.

Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we have found that alpha-globin pre-mRNA is stabilized by a mechanism that does not modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. We found an inverse relationship between mRNA levels and the length of the introduced fragments. In fact, mRNA levels as low as 1% were observed after inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment of 86 nt at the same position had no effect on mRNA accumulation. DNA insertion induced no change in transcription rate or in half-life of mature mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreased levels of nuclear pre-mRNA in proportion to that observed for mature mRNA. In contrast, the insertion of the 477-nt exogenous DNA in the last intron had no effect on mRNA levels despite the presence of intronic sequences in the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR. The fact that the half-life of mature mRNA is not affected by DNA insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation.

3' Untranslated Regions↗
PubMed1997

Expression of messenger ribonucleic acid (mRNA) encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles: identification of dominant follicles by expression of 3beta-HSD mRNA within the granulosa cell layer.

The objective of the present study was to examine changes in expression of mRNA encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) following estrus. Expression of 3beta-HSD mRNA was localized by in situ hybridization and quantified by image analysis. Expression of 3beta-HSD mRNA was first detected in theca interna cells of preantral follicles with a well-developed theca layer and in granulosa cells of follicles > or = 8 mm in diameter. Regardless of stage of follicular wave, expression of 3beta-HSD mRNA in granulosa cells of follicles > or = 8 mm was correlated with follicular size (r = 0.665; p < 0.01). The 36-h time period appeared to be a transition period for selection since dominant follicles were detected by size and expression of 3beta-HSD mRNA in some cows but not in others. By 48 h after wave initiation, dominant follicles could be identified by both size and expression of 3beta-HSD mRNA. Expression of mRNA for 3beta-HSD in theca cells was higher (p < 0.05) at 24 h than at 12 h and remained elevated thereafter through 96 h. In contrast to theca cells, expression of mRNA for 3beta-HSD was undetectable within granulosa cells at 12 and 24 h. At 36 h, 3beta-HSD mRNA was expressed in granulosa cells of healthy follicles > or = 8 mm, and expression was higher (p < 0.05) at 48 h compared with 36 h. Expression of 3beta-HSD mRNA levels increased further in granulosa cells (p < 0.05) at 84 and 96 h compared to 48 h. Upon detection of mRNA for 3beta-HSD in granulosa cells, high levels of expression were always found in one (dominant) follicle/cow with the exception of two cows at 36 and 84 h that expressed 3beta-HSD mRNA in two large healthy follicles. Expression of 3beta-HSD mRNA was also detectable in granulosa cells of a few large atretic follicles in which remnant granulosa cells appeared to be luteinized. Healthy follicles expressed higher (p < 0.05) levels of 3beta-HSD mRNA in both theca and granulosa cells than did atretic follicles. Expression of 3beta-HSD mRNA in theca cells was higher (p < 0.01) in dominant follicles than in other subordinate healthy follicles. These results indicate that only selected dominant follicles express 3beta-HSD mRNA within granulosa cells, and expression increased in both thecal and granulosa cells during the follicular wave. Therefore, expression of 3beta-HSD mRNA within granulosa cells may be associated with the mechanism of selection of the dominant follicle during a follicular wave and may be required for maximum steroid production during follicular dominance.

Animals↗
PubMed1992

Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA.

Influenza virus unspliced NS1 mRNA, like retroviral pre-mRNAs, is efficiently exported from the nucleus and translated in the cytoplasm of infected cells. With human immunodeficiency virus (HIV), the transport of viral pre-mRNAs is facilitated by the viral Rev protein. We tested the possibility that the influenza virus NS1 protein, a nuclear protein that is encoded by unspliced NS1 mRNA, has the same function as the HIV Rev protein. Surprisingly, using transient transfection assays, we found that rather than facilitating the nucleocytoplasmic transport of unspliced NS1 mRNA, the NS1 protein inhibited the transport of NS2 mRNA, the spliced mRNA generated from NS1 mRNA. The efficient transport of NS2 mRNA from the nucleus to the cytoplasm occurred only when the synthesis of the NS1 protein was abrogated by amber mutations. The NS1 protein down-regulated the export of NS2 mRNA whether or not it was generated by splicing, indicating that the NS1 protein acted directly on transport. Actinomycin D chase experiments verified that the NS1 protein acted on the transport and not on the differential stability of NS2 mRNA in the nucleus as compared to the cytoplasm. In addition, the NS1 protein inhibited the transport of NS1 mRNA itself, which contains all of the sequences in NS2 mRNA, particularly when NS1 mRNA was released from the splicing machinery by mutating its 3'-splice site. Our results indicate that the NS1 protein-mediated inhibition of transport requires sequences in NS2 mRNA. The transport of the viral PB1 protein, nucleocapsid protein, hemagglutinin, membrane protein, and M2 mRNAs was not affected by the NS1 protein. When the NS2 mRNA sequence was covalently attached to the PB1 mRNA, the transport of the chimeric mRNA was inhibited by the NS1 protein. Our results identify a novel function of the influenza virus NS1 protein and demonstrate that post-transcriptional control of gene expression can also occur at the level of the nucleocytoplasmic transport of a mature, spliced mRNA.

Amino Acid Sequence↗
PubMed1993

The glucagon-insulin antagonism in the regulation of cytosolic protein binding to the 3' end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes. Possible involvement in the stabilization of the mRNA.

Since protein binding to the 3' end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon-induced phosphoenolpyruvate-carboxykinase-mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24-h cultured rat hepatocytes to the 3' end of PCK mRNA. PCK-mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility-shift assay. In 24-h cultured rat hepatocytes, binding of cytosolic protein to the PCK-mRNA 3' end and PCK-mRNA levels were increased to a transient maximum at 2 h and 2-4 h, respectively, by a 1-nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon-induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin. 100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3' end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon-induced increase in PCK-mRNA levels by 30%. The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon-enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA. Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK-mRNA 3' end. The proximal 466 bases of the PCK-mRNA 3' end as well as the 1050 bases of the histone-H1(0)-mRNA 3' end and the 1200 bases of the arylsulfatase-A-mRNA 3' end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin. The 3' end of PCK, arylsulfatase A and H1(0) mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3' end of PCK mRNA and cytosolic extracts from liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.

Adenoviridae↗
PubMed1998

Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA.

The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.

3T3 Cells↗
PubMed1996

Control of apolipoprotein B mRNA editing: implication of mRNA dynamics at various maturation stages.

Apolipoprotein (apo) B mRNA editing is a genetic regulatory mechanism whereby nucleotide 6666 in apo-B-100 mRNA is converted from a C to a U. The end result of this change is the creation of a premature stop codon so that the translation product of the edited (apoB-48) mRNA contains 2152 amino acids residues instead of 4536 residues in the product (apoB-100) encoded by the unedited mRNA. ApoB mRNA editing is a post-transcriptional process that is expressed in a tissue-specific manner. Here we present a model for the control of apoB mRNA editing. The three variables in the model include editing activity, apoB mRNA transcription rate and mRNA degradation rate at various maturation stages for the mRNA, and a simple formula can be used to quantify the degree of apoB mRNA editing with respect to these variables at different apoB mRNA maturation stages. Time-dependent equations were solved numerically. Using this model, it can be shown that, in addition to editing activity, the degradation kinetics of apoB mRNA can also serve as an efficient modulator of the degree of editing. The rate of apoB mRNA transcription has a transient effect on the degree of editing; varying the apoB mRNA degradation constant tends to change the degree of editing only at the specific maturation stage. This model can render some previously proposed hypothesis (e.g. coupling of mRNA editing to splicing/polyadenylation) unnecessary and provides a rational basis for the design of experiments on specific aspects of apoB mRNA editing in the future; it may also be applicable to other types of RNA editing.

Animals↗
PubMed1997

Decreased Stability of Transforming Growth Factor beta Type II Receptor mRNA in RER+ Human Colon Carcinoma Cells

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.

Journal Article↗
PubMed2002

AUUUA motifs in the 3'UTR of human glucocorticoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor protein expression.

An association between a gene polymorphism of the human glucocorticoid receptor (hGR) gene and rheumatoid arthritis has recently been suggested. This polymorphism contains an A to G mutation in the 3'UTR of exon 9beta, which encodes the 3'UTR of the mRNA of the hGRbeta isoform. The hGRbeta isoform can act as a dominant negative inhibitor of hGRalpha, and therefore may contribute to glucocorticoid resistance. The A to G mutation is located in an AUUUA motif, which is known to destabilize mRNA. In the present study, the importance of the mutation in this AUUUA motif was further characterized and mutations in other AUUUA motifs in the 3'UTR of hGRbeta and hGRalpha mRNA were studied. hGRbeta and hGRalpha expression vectors, carrying mutations in one AUUUA motif or all AUUUA motifs were transiently transfected into COS-1 cells. Each transfected vector was analyzed for the mRNA expression level, the mRNA turnover rate and the protein expression level. The naturally occurring mutation in the 3'UTR of hGRbeta mRNA increased mRNA stability and protein expression. Mutation of two other AUUUA motifs in the 3'UTR of hGRbeta, or mutation of all four AUUUA motifs resulted in a similar effect. Mutation of the most 5' AUUUA motif did not alter hGRbeta mRNA expression or mRNA stability. Mutation of all 10 AUUUA motifs in the 3'UTR of hGRalpha mRNA increased hGRalpha mRNA expression and mRNA stability as well as expression of the receptor protein level. Thus, the naturally occurring mutation in an AUUUA motif in the 3'UTR of hGRbeta mRNA results not only in increased mRNA stability, but also in increased receptor protein expression, which may contribute to glucocorticoid resistance. A similar role is suggested for two other AUUUA motifs in the 3'UTR of hGRbeta mRNA and for the 10 AUUUA motifs that are present in the 3'UTR of hGRalpha.

3' Untranslated Regions↗
PubMed1994

Cross-linking of mRNA analogues containing 4-thiouridine residues on the 3'- or 5'-side of the coding triplet to the mRNA binding center of the human ribosome.

The interaction between mRNA and 18S rRNA within complexes of human placenta 80S ribosomes has been investigated by photochemical cross-linking experiments using mRNA analogues substituted with 4-thiouridine at specific locations. mRNA analogues 51 or 54 nucleotides long were prepared from synthetic DNA templates. These mRNA analogues contained either the sequence GGGACC (coding for glycine and threonine, respectively) or the single triplet GGG together with 2-4 4-thiouridine residues located at various positions with respect to the coding triplets. The products of cross-linking of the mRNA analogues to 18S rRNA within different model complexes without tRNA or in the presence of cognate tRNAs were analyzed by reverse transcription. Two cross-linking sites in the 18S rRNA were detected. The first site, U630, was cross-linked by mRNA 8' (s4U at +20, +22, +24, and +26), mRNA 9e' (s4U at -16, -18, and -20), and mRNA 10 (s4U at +4, +6, -1, and -3) but, unexpectedly, not with either mRNA 10b (s4U at +4 and +6) or mRNA 10c (s4U at -1 and -3). The second site, U1111/A1112, was cross-linked by mRNA 10 and mRNA 10c but not by any of the other mRNA analogues. There is significant tRNA dependence on cross-linking only for mRNA analogue 9e'. Both of the sites detected correspond to sites of mRNA cross-linking in Escherichia coli 16S rRNA.

Base Sequence↗
PubMed2004

Detection of thyrotropin-receptor messenger ribonucleic acid (mRNA) and thyroglobulin mRNA transcripts in peripheral blood of patients with thyroid disease: sensitive and specific markers for thyroid cancer.

Because thyroid cancer cells express functional TSH receptors (TSHR), TSHR-mRNA in peripheral blood might serve as a tissue-/cancer-specific marker. We measured circulating TSHR-mRNA by RT-PCR in 51 normal controls, 27 patients with benign thyroid disease, 67 patients with treated differentiated thyroid cancer (DTC), and eight patients with newly diagnosed DTC, preoperatively. Results were compared with thyroglobulin (Tg) mRNA and serum Tg levels. TSHR-mRNA signals were not detected in normal controls and in 24 of 27 (89%) patients with benign thyroid disease. All 19 patients with treated DTC with evidence of distant or local disease tested positive for TSHR-mRNA (sensitivity 100%). Among patients with no evidence of disease, TSHR-mRNA was detected in 1 in 48 (specificity 98%). Six of the eight newly diagnosed DTC patients tested preoperatively were positive for TSHR-mRNA. The concordance between TSHR-mRNA and Tg-mRNA and between TSHR-mRNA and serum Tg was 95%. Fourteen patients with DTC (21%) had Tg antibodies, three with local disease (all positive for TSHR-mRNA), and 11 with no evidence of disease (all negative for TSHR-mRNA). Our results indicate that TSHR-mRNA and/or Tg-mRNA in peripheral blood are both equally sensitive and specific markers for monitoring thyroid cancer patients. Their principal value resides in the Tg antibody-positive patients in whom a positive or a negative mRNA value might have indicated or obviated the need for a whole-body scan. Furthermore, the high specificity combined with their ability to predict thyroid cancer preoperatively suggests a potential role in detecting thyroid cancer in patients with thyroid nodules.

Adult↗
PubMed2005

Total cyclooxygenase-2 mRNA levels correlate with vascular endothelial growth factor mRNA levels, tumor angiogenesis and prognosis in non-small cell lung cancer patients.

Interaction between cancer cells and adjacent stromal cells is important to promote tumor development. Our aim was to study total COX-2 mRNA expression in both cancer cells and surrounding stromal cells and its association with angiogenic factor VEGF mRNA expression, tumor angiogenesis and prognosis in patients with NSCLC. COX-2 mRNA expression in both cancer cells and stromal tissue was analyzed using real-time quantitative (RTQ) RT-PCR in 60 NSCLC surgical specimens. Immunohistochemistry (IHC) was used to localize COX-2 protein in tumor specimens. Correlations between tumoral total COX-2 mRNA expression and VEGF mRNA expression (measured by RTQ RT-PCR), intratumoral microvessel counts (evaluated by IHC), other clinicopathologic variables, survival and relapse were tested. COX-2 protein expression was found in cancer as well as the surrounding stromal cells (including infiltrating inflammatory cells and endothelial cells of tumor-associated microvessels). VEGF protein expression was mainly located in cancer cells. There was a significant association between high tumoral total COX-2 mRNA expression and high VEGF mRNA expression (p = 0.01) or high intratumoral MVC (p < 0.001) but not other clinicopathologic variables, including tumor status and lymph node metastasis. Patients with higher tumoral total COX-2 mRNA expression had a statistically shorter survival time (median 15.0 +/- 2.61 months) and relapse time (median 5.0 +/- 1.37 months) than those with lower tumoral total COX-2 mRNA expression (median 40.0 +/- 3.12 and 34.0 +/- 3.11 months; p < 0.0001 and p < 0.0001, respectively, log-rank test). A significant difference in survival and relapse time was also seen between patients with high and low tumoral VEGF mRNA expression and between those with high and low intratumoral MVC (p = 0.0046 and p = 0.0038, respectively). After stratification by disease stage or histologic subtype, the prognostic significance of high total COX-2 mRNA expression was still apparent in both stage I and stage II-IV and in both squamous cell carcinoma and adenocarcinoma (p < or = 0.01 for all). Multivariate analysis using the Cox regression model with backward elimination showed that tumoral total COX-2 mRNA expression and lymph node status were the 2 most important independent prognostic predictors for survival and disease relapse. We report that total COX-2 mRNA expression in cancer cells and surrounding stromal cells correlates strongly and positively with VEGF mRNA expression, intratumoral MVC and adverse prognosis in NSCLC patients. This implies that COX-2 expression in both cancer cells and stromal cells within the tumor microenvironment may play an important role in upregulating the expression of the angiogenic factor VEGF and tumor angiogenesis in NSCLC and explains, in part, the adverse prognostic effect of COX-2 overexpression in patients with NSCLC.

Adenocarcinoma↗
PubMed1993

Regulation of anterior pituitary gonadotropin subunit mRNA levels during the preovulatory gonadotropin surge: a physiological role of progesterone in regulating LH-beta and FSH-beta mRNA levels.

In a previous study we demonstrated that in the ovariectomized estrogen-primed immature rat, progesterone induced a gonadotropin surge while the gonadotropin mRNA subunit levels were either suppressed or unaltered. This observation has now been confirmed using more frequent time points. Progesterone administered at 0900 h was found to suppress LH-beta mRNA levels at 1300, 1400, and 0800 h the next day, with no subsequent effects at 1000, 1200 or 1600 h. FSH-beta mRNA levels were unaffected by progesterone except for a slight elevation at 1400 h and a suppression at 0800 h. Progesterone was either suppressive or had no effect on alpha mRNA levels. Since elevations in LH-beta and FSH-beta mRNA levels were observed in the cycling rat, the observed differences in the ovariectomized estrogen-primed rat could be due to a higher basal synthesis occurring due to ovariectomy. This was indeed the case because LH-beta and FSH-beta mRNA levels were 3.7- and 42.7-fold higher in such animals as compared to intact estrogen-primed rats. In contrast to the ovariectomized estrogen-primed rats, in intact estrogen-primed rats LH-beta mRNA levels were increased at 1000 h and FSH-beta mRNA levels were increased at 1000, 1200 and 1300 h after the administration of progesterone. In pregnant mare's serum gonadotropin-primed immature rats, LH-beta, FSH-beta and alpha-subunit mRNA levels were significantly elevated at 1800 and 2000 h, paralleling the serum LH and FSH surge. The progesterone antagonist RU486 (0.2 and 1.0 mg) significantly reduced serum LH and FSH levels at 2000 h. The lower dose reduced LH-beta and alpha-subunit mRNA levels at 2000 h and FSH-beta mRNA levels at 1800 h. The higher dose caused an increase in LH-beta mRNA levels at 1200 and 1800 h and a decrease in FSH-beta mRNA levels at 1800 and 2000 h. In conclusion, the present study provides evidence that preovulatory progesterone plays an important role in the increase in FSH-beta mRNA levels as well as the release of LH and FSH during the normal preovulatory gonadotropin surge. This relationship appears to be dependent on the ongoing rate of synthesis because this does not occur in the ovariectomized estrogen-primed rat in which synthesis is at a high basal level. Furthermore, the correlation with FSH appears to be tighter as compared to LH.

Animals↗
PubMed1999

Dimorphic expression of medial basal hypothalamic-preoptic area calbindin-D(28K) mRNA during perinatal development and adult distribution of calbindin-D(28K) mRNA in Sprague-Dawley rats.

The calcium-binding protein, calbindin (CALB) is: (a) distributed throughout the central nervous system (CNS), (b) abundant in neurons and, (c) thought to act as a buffer by binding intracellular calcium, mediating neurogenesis (cell profileration) and neuronal programmed cell death. Using Northern analysis, CALB mRNA distribution was characterized in 12 different adult brain regions. Additionally, CALB mRNA levels were characterized in the medial basal hypothalamus (MBH) and preoptic area (POA) in perinatal male and female rats, in order to compare this mRNA pattern to the dimorphic MBH-POA CALB protein profile our laboratory previously reported. Three CALB mRNA species were detected (at 1.9, 2.8 and 3.2 kilobase pairs) in all CNS regions. The smallest mRNA transcript (at 1.9 kilobase pairs) was the most abundant of the three CALB mRNAs. To quantify these mRNA signals, CALB mRNA levels were normalized to 18s ribosomal RNA bands. Among the various adult brain sites assayed, the cerebellum expressed the highest CALB mRNA signals. High CALB mRNAs were observed in the MBH-POA, olfactory bulb and hippocampal regions. Moderate CALB mRNA levels were seen in the striatum and frontal cortex while moderate to low CALB mRNA levels were observed in the posterior cortex, entorhinal cortex, midbrain, pons, thalamus and medulla. During perinatal development, MBH-POA CALB mRNA levels were lowest at gestation day (GD) 18, increased four-fold in newborns and remained at moderate levels during early postnatal development. Male CALB mRNA levels were notably greater than female values at GD 18 and in newborns. Whereas, at PND 2, the CALB mRNA levels were approximately equivalent in males and females. These findings suggest that in the adult CNS CALB mRNAs vary among different brain regions. The present male vs. female MBH-POA CALB mRNA levels confirm previously reported dimorphic protein patterns of MBH-POA CALB during perinatal development. Thus, the genesis of sexually dimorphic structures may be influenced by the dimorphic CALB expression in the MBH-POA region.

Animals↗
PubMed2006

Essential role of 3'-untranslated region-mediated mRNA decay in circadian oscillations of mouse Period3 mRNA.

Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3'-UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3'-UTR-mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations.

3' Untranslated Regions↗
PubMed1981

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, "P." We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.

Animals↗
PubMed1995

Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels.

Hypothalamic-pituitary-adrenal (HPA) responses remain intact or increase after chronic or repeated stress despite robust levels of circulating glucocorticoids that would be expected to restrain the responsiveness of the axis. The purpose of this study was to determine whether chronic stress altered corticosteroid receptor messenger RNA (mRNA) levels at any locus known to mediate glucocorticoid feedback on HPA function (i.e. hippocampus or hypothalamus), whether such effects were glucocorticoid dependent, and whether changes in corticosteroid receptor function could potentially contribute to the putative shift from corticotropin-releasing hormone (CRH) to arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) in the modulation of pituitary adrenal function occurring during chronic stress. We compared the stress responsiveness of sham-operated rats to that of adrenalectomized rats using a moderate dose of corticosterone (CORT) pellet replacement (ADX + CORT group). Acute immobilization caused a significant increase in CRH, but not AVP, mRNA levels in the parvocellular PVN in sham rats. The ADX + CORT group showed significantly greater increases in both CRH and AVP mRNA levels in the PVN compared to sham rats. These data indicate that PVN AVP mRNA levels are more sensitive to glucocorticoid negative feedback than are the levels of CRH mRNA. In repeated stress, the sham groups showed robust increases in PVN CRH and AVP mRNA levels despite high levels of plasma CORT. The rise in AVP mRNA levels was greater than that in CRH mRNA. Type II glucocorticoid receptor mRNA in the hippocampus and PVN was decreased in the repeatedly stressed sham group. These data suggest a decrease in the CORT negative feedback restraint of PVN CRH and AVP mRNA levels repeated stress and a persistence of relatively greater responsiveness of AVP mRNA levels to CORT negative feedback. After repeated stress in ADX+CORT rats, both PVN CRH and AVP mRNA levels showed robust responses, with a relatively greater increase in AVP mRNA. These data indicate that a CORT-mediated decrease in hippocampal and hypothalamic glucocorticoid receptor mRNA levels is not the only mechanism contributing to the maintenance of a robust HPA response after repeated stress. Similarly, we postulate that the relative shift from CRH to AVP in the PVN after repeated stress is mediated by both a greater sensitivity of AVP to CORT negative feedback and CORT-independent mechanisms.

Adrenalectomy↗
PubMed2006

Correlative studies on uPA mRNA and uPAR mRNA expression with vascular endothelial growth factor, microvessel density, progression and survival time of patients with gastric cancer.

AIM: To investigate the correlations between the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and vascular endothelial growth factor (VEGF) protein and clinicopathologic features, microvessel density (MVD) and survival time. METHODS: In situ hybridization and immuno-histochemistry techniques were used to study the expressions of uPA mRNA, uPAR mRNA, VEGF and CD34 protein in 105 gastric carcinoma specimens. RESULTS: Expressions of uPA mRNA, uPAR mRNA and VEGF protein were observed in 61 (58.1%) cases, 70 (66.7%) cases and 67 (63.8%) cases, respectively. The uPA mRNA and uPAR mRNA positive expression rates in infiltrating-type cases (73.7%, 75.4%), stage III-IV (72.1%, 75.4%), vessel invasion (63.2%, 69.9%), lymphatic metastasis (67.1%, 74.4%) and distant metastasis (88.1%, 85.7%) were significantly higher than those of the expanding-type (chi2 = 15.57, P = 0.001; chi2 = 6.91, P = 0.046), stage I-II (chi2 = 19.22, P = 0.001; chi2 = 16.75, P = 0.001), non-vessel invasion (chi2 = 11.92, P = 0.006; chi2 = 14.15, P = 0.002), non-lymphatic metastasis (chi2 = 28.41, P = 0.001; chi2 = 22.5, P = 0.005) and non-distant metastasis (chi2 = 12.32, P = 0.004; chi2 = 17.42, P = 0.002; chi2 = 11.25, P = 0.012; chi2 = 18.12, P = 0.002). The VEGF positive expression rates in infiltrating-type cases (75.4%), stage III-IV (88.5%), vessel invasion (82.9%), lymphatic metastasis (84.3%) and distant metastasis (95.2%) were significantly higher than those of the expanding-type (chi2 = 9.61, P = 0.021), stage I-II (chi2 = 16.66, P = 0.001), non-vessel invasion (chi2 = 29.38, P = 0.001), non-lymphatic metastasis (chi2 = 18.68, P = 0.005), and non-distant metastasis (chi2 = 22.72, P = 0.007; chi2 = 21.62, P = 0.004). The mean MVD in the specimens positive for the uPA mRNA, uPAR mRNA and VEGF protein was markedly higher than those with negative expression groups. Moreover, a positive relation between MVD and uPA mRNA (rs = 0.199, P = 0.042), uPAR mRNA (rs = 0.278, P = 0.035), and VEGF (rs = 0.398, P = 0.048) expressions was observed. The mean survival time in cases with positive uPA mRNA, uPAR mRNA and VEGF protein expression or MVD value > or = 54.9 was significantly shorter than those in cases with negative expression or MVD value < 54.9. CONCLUSION: uPA and uPAR expressions are correlated with enhanced VEGF-induced tumor angiogenesis and may play a role in invasion and nodal metastasis of gastric carcinoma, thereby serving as prognostic markers of gastric cancer.

Adenocarcinoma↗