No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Posttranscriptional Regulation of Thyrotropin Subunit Messenger Ribonucleic Acid by Thyroid Hormone in Murine Thyrotrope Tumor Cells: A Conserved Mechanism across Species
Abstract
Thyroid hormone (T3) negatively regulates TSH b-subunit (TSHb) messenger RNA (mRNA) gene expression in whole rat pituitary, in part at the level of mRNA stability. However, the regulation of TSHb mRNA turnover by T3 in pure populations of thyrotropes and in other species is unknown. To further investigate this, we used murine thyrotropic TtT97 tumor cells. Using primary cultures of TtT97 cells, T3 down-regulated TSHb mRNA to ;35% of the control level by 8 h. Actinomycin D chase revealed that T3 destabilized TSHb mRNA, reducing the half-life from ;24 to 7 h, and was accompanied by a decrease in TSHb mRNA size. Ribonuclease H analysis revealed that this T3-induced decrease in size was due to a shortening of poly(A) tail from ;160 to ;30 nucleotides and was specific for TSHb mRNA. Cycloheximide mimicked the poly(A) tail effect observed with T3 .I n the absence of T3, actinomycin D deadenylated TSHb mRNA without inducing rapid decay. We conclude that T3 reduces the steady state half-life of TSHb mRNA in murine TtT97 thyrotropic tumor cells accompanied by a reduction in poly(A) tail length. However, in the absence of T3, deadenylation alone is not sufficient to induce TSHb mRNA decay. Together with the high degree of sequence conservation in the 39-untranslated region of murine and rat TSHb mRNA se- quences and the similarities of the T3 effect, these data provide the first evidence for a highly conserved posttranscriptional mechanism operative across species. We propose a model in which T3 coordinately regulates shortening of the poly(A) tail and the activity of a trans- acting RNA-binding protein and/or an exonuclease to accelerate TSHb mRNA turnover. (Endocrinology 139: 1093-1100, 1998)
... We and others have shown that TSH subunit mRNA is regulated posttranscriptionally by T 3 , reducing its half-life from 24 h to approximately 8 h in both rat and murine pituitary cells (Krane et al. 1991, Staton & Leedman 1998. This reduction in mRNA stability was accompanied in both species with a reduction in the length of the poly(A) tail from 160-180 to 30 nt. ...
... Several studies have shown that T 3 decreases TSH -and TSH -subunit steady-state mRNA levels, in part as a result of reduced transcription (Surks & Litschitz 1977, Ross et al. 1983, Shupnik et al. 1985. TSH mRNA is also regulated by T 3 at the posttranscriptional level, accompanied by a shortening of the poly(A) tail (Krane et al. 1991, Staton & Leedman 1998). In the presence of actinomycin D, but the absence of T 3 , TSH mRNA was deadenylated without inducing rapid mRNA decay, suggesting that deadenylation alone was not sufficient for the acceleration of TSH mRNA turnover. ...
... We recently isolated a cis-acting element located within the 70 nt of the 3 UTR of TSH mRNA (unpublished observations). This sequence does not 2. Summary of pituitary-derived genes that are known to be regulated by hormones Staton & Leedman (1998) mRNA destabilized twofold by bromocryptine Levy & Lightman (1990) contain any AREs or other well characterized cis-acting destabilizing elements (Leedman et al. 1995). Interestingly, it was the target for T 3regulated cytoplasmic RNA-binding proteins derived from murine thyrotrope cells. ...
Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.
... In a separate study, estrogen was shown to increase the mRNA stability of another pituitary hormone receptor, TRHR, approximately twofold in rat pituitary primary cultures (Kimura et al. 1994). We and others have shown that TSH subunit mRNA is regulated posttranscriptionally by T 3 , reducing its half-life from 24 h to approximately 8 h in both rat and murine pituitary cells (Krane et al. 1991, Staton & Leedman 1998). This reduction in mRNA stability was accompanied in both species with a reduction in the length of the poly(A) tail from 160–180 to 30 nt. ...
... Several studies have shown that T 3 decreases TSH-and TSH-subunit steady-state mRNA levels, in part as a result of reduced transcription (Surks & Litschitz 1977, Ross et al. 1983, Shupnik et al. 1985). TSH mRNA is also regulated by T 3 at the posttranscriptional level, accompanied by a shortening of the poly(A) tail (Krane et al. 1991, Staton & Leedman 1998). In the presence of actinomycin D, but the absence of T 3 , TSH mRNA was deadenylated without inducing rapid mRNA decay, suggesting that deadenylation alone was not sufficient for the acceleration of TSH mRNA turnover. ...
... mRNA affected TRHR mRNA stabilized twofold by estrogen Kimura et al. (1994) TSH mRNA destabilized three-to fourfold and poly(A) tail shortened by T 3 ; Staton & Leedman (1998) mRNA mRNA decay in the pituitary · and otherscontain any AREs or other well characterized cis-acting destabilizing elements (Leedman et al. 1995 ). Interestingly, it was the target for T 3 regulated cytoplasmic RNA-binding proteins derived from murine thyrotrope cells. ...
Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.
... Further, O-GlcNAcylation competes with phosphorylation sites on oncogenes and tumor suppressors to increase proliferation and promote invasion, making it an essential mediator in tumor formation and progression (20)(21)(22)(23) promoter activation consequences of the CRHdownstream signaling pathway. Interestingly, this mechanism exists for many hormones, including TSH, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) mRNA (31)(32)(33)(34)(35)(36), and has been suggested for POMC pre-hormone as well (37). Furthermore, O-GlcNAcylation is essential for this novel regulatory pathway as it enhances the proper splicing of Pomc mRNA and prevents its decay. ...
... Furthermore, we demonstrate, for the first time, that CRH induction can mitigate this decay, quickly resulting in a stabilization of Pomc mRNA in an O-GlcNAc-dependent manner (Figure 7). While mRNA stabilization mechanisms have been studied for other pituitary hormones, the regulatory proteins involved in Pomc mRNA degradation remain unknown (31)(32)(33)35,36,(85)(86)(87). In light of our O-GlcNAcomic analysis, intron retention and associated degradation by nonsensemediated decay appeared to be the most plausible mechanism for this rapid change in Pomc mRNA stability. ...
Pituitary adenomas have a staggering 16.7% lifetime prevalence and can be devastating in many patients due to profound endocrine and neurologic dysfunction. To date, no clear genomic or epigenomic markers correlates with their onset or severity. Herein, we investigate the impact of the O -GlcNAc post-translational modification in their etiology. Found in over 5000 human proteins to date, O -GlcNAcylation dynamically regulates proteins in critical signaling pathways, and its deregulation is involved in cancers progression and endocrine diseases such as diabetes.
In this study, we demonstrate that O -GlcNAcylation enzymes were upregulated, particularly in aggressive ACTH-secreting tumors, suggesting a role for O -GlcNAcylation in pituitary adenoma etiology. In addition to the demonstration that O -GlcNAcylation was essential for their proliferation, we show that the endocrine function of pituitary adenoma is also dependent on O -GlcNAcylation. In corticotropic tumors, hyper-secretion of the proopiomelanocortin (POMC)-derived hormone ACTH leads to Cushing’s disease, materialized by severe endocrine disruption and increased mortality. We demonstrate that Pomc mRNA is stabilized in an O -GlcNAc-dependent manner in response to corticotropic-stimulating hormone (CRH). By impacting Pomc mRNA splicing and stability, O -GlcNAcylation contributes to this new mechanism of fast hormonal response in corticotropes. Thus, this study stresses the essential role of O -GlcNAcylation in ACTH-secreting adenomas’ pathophysiology, including cellular proliferation and hypersecretion.
... Die mRNA-Stabilität (Halbwertszeit, turn-over) kann einen grossen Einfluss auf die Expression individueller Gene in der Zelle haben, denn die Abbauraten verschiedener mRNA´s variieren sehr stark mit bis zu 100 fachen Unterschieden in der biologischen Halbwertszeit von ~15 min (einige Onkogene wie z.B. myc, fos) bis zu 24 Stunden (Beelman and Parker, 1995;Jacobson and Peltz, 1996). Die Regulation des mRNA-Abbaus ist somit ein wichtiger Mechanismus für die Regulation der Genexpression (Li et al., 1997;Wilson et al, 1997;Ing and Ott, 1999;Staton and Leedman, 1998;Bourgeois et al., 1997). ...
... Wenn dieses AUreiche Sequenzmotiv in andere 3´-UTR´s, wie z.B. in die Globulin-mRNA eingefügt wird, die normalerweise sehr stabil sind, werden diese mRNA´s bis zu 30 mal schneller abgebaut (Shaw and Kamen, 1986). Darüber hinaus können manche Hormone die Genexpression dadurch beeinflussen, daß sie die Modifikation des RBPs induzieren und die Bindungsaffinität an die mRNA ändern (Staton and Leedman, 1998;Ing and Ott, 1999). ...
Humanes Cathepsin L (hCATL) ist eine lysosomale Cystein-Protease und bei der Tumor-Invasion und -Metastasierung beteiligt. Die Expression des hCATL korreliert in vielen normalen Geweben und Tumorgeweben positiv mit der mRNA-Expression. Eine Ausnahme stellt das Nierenkarzinom dar, wo trotz erhöhter mRNA-Expression der hCATL-Proteingehalt erniedrigt ist. Um dieses Missverhältnis in malignen Nierentumoren gegenüber gesundem Nierengewebe zu klären, wurden in der vorliegender Arbeit die Spleissvarianten der hCATL-mRNA kloniert und charakterisiert.
... These actions include alterations in Ca 2ϩ , Na ϩ , and glucose transport through the plasma membrane, protein-kinases activity, translation efficiency, and half-life of specific mRNAs and mitochondrial respiration (11-15). These data and other accumulating evidences indicate that, besides its well-characterized genomic actions, TH also acts at the posttranscriptional level, the mechanisms of which are still not well understood (15)(16)(17)(18)(19). ...
... These actions include alterations in Ca 2ϩ , Na ϩ , and glucose transport through the plasma membrane, protein-kinases activity, translation efficiency, and half-life of specific mRNAs and mitochondrial respiration (11)(12)(13)(14)(15). These data and other accumulating evidences indicate that, besides its well-characterized genomic actions, TH also acts at the posttranscriptional level, the mechanisms of which are still not well understood (15)(16)(17)(18)(19). ...
Thyroid hormone was shown to induce actin cytoskeleton polymerization in hypothyroid astrocytes and osteoblastic cells by a nongenomic mechanism. Polyadenylation of GH mRNA, a process that depends on cytoskeleton-associated proteins, was also shown to be regulated by thyroid hormone. Here we investigated by histochemistry and immunohistochemistry whether acute (100 microg per 100 g body weight, iv, for 30 min) or chronic (5 microg per 100 g body weight, ip, 5 d) administration of T3 to thyroidectomized (Tx) and sham-operated rats affects the somatotrophs F-actin cytoskeleton arrangement and its potential repercussion on GH synthesis and secretion. Thyroidectomy dramatically decreased the amount of somatotrophs F-actin content and induced the disassembly of the actin cytoskeleton. These effects were reversed by acute and chronic administration of T3. In addition, in Tx rat somatotrophs, GH labeling was detected mostly at the cell periphery. After 30 min of T3 administration, GH labeling decreased at periphery and increased in the perinuclear region, suggesting that GH secretion and synthesis were stimulated by T3. No differences were detected in the total actin protein content, although a decrease in the F- and increase in G-actin contents were detected in Tx rat pituitaries, a panorama that was reversed by acute T3 treatment, as shown by Western blotting analysis. The sham-operated animals' somatotrophs were only mildly affected by acute T3 administration. The results indicate that the T3-induced rapid alterations on somatotroph actin cytoskeleton and GH cellular distribution resulted from actin filaments rearrangement, which characterizes a nongenomic action.
... We, therefore, hypothesize that O-GlcNAcylation contributes to pituitary tumor development. More precisely, we believe that O- mRNA (33)(34)(35)(36)(37)(38), and has been suggested for POMC pre-hormone as well (39). Furthermore, O-GlcNAcylation is essential for this novel regulatory pathway as it enhances the proper splicing of Pomc mRNA and prevents its decay. ...
Pituitary adenomas have a staggering 16.7% lifetime prevalence and can be devastating in many patients due to profound endocrine and neurologic dysfunction. To date, no clear genomic or epigenomic markers correlates with their onset or severity. Herein, we investigate the impact of the O-GlcNAc post-translational modification in their etiology. Found in over 5000 human proteins to date, O-GlcNAcylation dynamically regulates proteins in critical signaling pathways, and its deregulation is involved in cancers progression and endocrine diseases such as diabetes.
In this study, we demonstrate that O-GlcNAcylation enzymes were upregulated, particularly in aggressive ACTH-secreting tumors, suggesting a role for O-GlcNAcylation in pituitary adenoma etiology. In addition to the demonstration that O-GlcNAcylation was essential for their proliferation, we show that the endocrine function of pituitary adenoma is also dependent on O-GlcNAcylation. In corticotropic tumors, hyper-secretion of the proopiomelanocortin (POMC)-derived hormone ACTH leads to Cushing’s disease, materialized by severe endocrine disruption and increased mortality. We demonstrate that Pomc mRNA is stabilized in an O-GlcNAc-dependent manner in response to corticotrophin-releasing hormone (CRH). By impacting Pomc mRNA splicing and stability, O-GlcNAcylation contributes to this new mechanism of fast hormonal response in corticotropes. Thus, this study stresses the essential role of O-GlcNAcylation in ACTH-secreting adenomas’ pathophysiology, including cellular proliferation and hypersecretion.
... 310Using Tx rats, we observed that in hypothyroidism the poly(A) tail length of Tshb 311 mRNA is increased, followed by a significant rise in the translational rate of Tshb, besides 312 the expected increase in the transcriptional process (Bargi-Souza et al. 2013). Thirty 313 minutes after treatment with T3, the poly(A) tail length of Tshb mRNA of Tx rats was 314 reduced to values similar to those observed in the poly(A) tail length of euthyroid rats and 315 was positively correlated to a marked reduction in the content of Tshb in polysome 316 fractions indicating that T3 rapidly reduced mRNA translation and TSH protein synthesis 317 (Bargi-Souza et al. 2013;Goulart-Silva et al. 2011).Previous studies regarding T3 effects on the Tshb mRNA poly(A) tail length have 319 already shown a decrease of its polyadenylation degree in hypothyroid pituitary cell 320 cultured with T3 for 8 hours and, this reduction was more pronounced after 24 hours 321 (Krane, et al. 1991;Staton and Leedman 1998). Euthyroid animals treated for 4 h with 322 higher doses of T3 also presented a reduction of Tshb mRNA poly(A) tail length (Krane et 323 al. 1991). ...
Thyroid hormones (TH) classically regulate gene expression by transcriptional mechanisms. In pituitary, the encoding genes for Growth Hormone (GH) and Thyroid Stimulating Hormone (TSH) are examples of genes regulated by triiodothyronine (T3) in a positive and negative way, respectively. Recent studies have shown a rapid adjustment of GH and TSH synthesis/secretion induced by T3 posttranscriptional actions. In somatotrophs, T3 promotes an increase in Gh mRNA content, poly(A) tail length and binding to the ribosome, associated with a rearrangement of actin cytoskeleton. In thyrotrophs, T3 reduces Tshb mRNA content, poly(A) tail length and its association to the ribosome. In parallel, it promotes a redistribution of TSH secretory granules to more distal regions of the cell periphery, indicating a rapid effect of T3 inhibition of TSH secretion. T3 was shown to affect the content of tubulin and the polymerization of actin and tubulin cytoskeletons in the whole anterior pituitary gland, and to increase intracellular alpha (CGA) content. This review summarizes genomic and non-genomic/posttranscriptional actions of TH on the regulation of several steps of GH and TSH synthesis and secretion. These distinct mechanisms induced by T3 can occur simultaneously, even though non-genomic effects are promptly elicited and precede the genomic actions, coexisting in a functional network within the cells.
... In contrast, mRNAs for proteins that are transiently expressed in response to a specific stimulus such as developmental, nutritional, hormonal, or environmental input, are often unstable, which enables the cell to downregulate translation rapidly. Examples of such unstable transcripts are mRNAs for growth factors, proto-oncogenes, cytokines, and lymphokines (Schiavi et al., 1994;Staton and Leedman, 1998). Apart from regulating the expression of an individual protein, mRNA turnover is a major control mechanism to synchronize gene expression. ...
... Considering that this effect was achieved in a short period of time (5 or 10 min) and that there is no consensus sequence for TH receptor binding in the AT1R gene promoter, we hypothesize that this action could occur as a consequence of posttranscriptional effects. In this sense, besides regulating the transcription of several genes, it has been found that THs may also have posttranscriptional effects on the stability of several mRNAs (Poddar et al. 1996, Staton & Leedman 1998, Kamegai et al. 2001, Liu & Waxman 2002, Danzi & Klein 2005, Minamisawa et al. 2006, Silva et al. 2010 ). One of the posttranscriptional mechanisms that influence the stability of mRNA is the increase in mRNA poly-A tail length, which protects the mRNA from degradation by RNases (Salles et al. 1999). ...
The angiotensin II type 1 receptor (AT1R) is involved in the development of cardiac hypertrophy promoted by thyroid hormone. Recently, we demonstrated that triiodothyronine (T₃) rapidly increases AT1R mRNA and protein levels in cardiomyocyte cultures. However, the molecular mechanisms responsible for these rapid events are not yet known. In this study, we investigated the T₃ effect on AT1R mRNA polyadenylation in cultured cardiomyocytes as well as on the expression of microRNA-350 (miR-350), which targets AT1R mRNA. The transcriptional and translational actions mediated by T₃ on AT1R levels were also assessed. The total content of ubiquitinated proteins in cardiomyocytes treated with T₃ was investigated. Our data confirmed that T₃ rapidly raised AT1R mRNA and protein levels, as assessed by real-time PCR and western blotting respectively. The use of inhibitors of mRNA and protein synthesis prevented the rapid increase in AT1R protein levels mediated by T₃. In addition, T₃ rapidly increased the poly-A tail length of the AT1R mRNA, as determined by rapid amplification of cDNA ends poly-A test, and decreased the content of ubiquitinated proteins in cardiomyocytes. On the other hand, T₃ treatment increased miR-350 expression. In parallel with its transcriptional and translational effects on the AT1R, T₃ exerted a rapid posttranscriptional action on AT1R mRNA polyadenylation, which might be contributing to increase transcript stability, as well as on translational efficiency, resulting to the rapid increase in AT1R mRNA expression and protein levels. Finally, these results show, for the first time, that T₃ rapidly triggers distinct mechanisms, which might contribute to the regulation of AT1R levels in cardiomyocytes.
... Staton at al. postulated that such a mechanism is highly conserved across the species. In addition to previous studies they observed that simple deadenylation of TSH messenger body is insufficient to induce its decay [31]. This suggests the triggering of additional mechanism such as exonuclease digestion and actions of other still not identified mRNA binding proteins. ...
ABSTRACT: RNA-binding proteins may regulate every aspect of RNA metabolism, including pre-mRNA splicing, mRNA trafficking, stability and translation of many genes. The dynamic association of these proteins with RNA defines the lifetime, cellular localization, processing and the rate at which a specific mRNA is translated. One of the pathways involved in regulating of mRNA stability is mediated by adenylate uridylate-rich element (ARE) binding proteins. These proteins are involved in processes of apoptosis, tumorigenesis and development. Out of many ARE-binding proteins, two of them AUF1 and HuR were studied most extensively and reported to regulate the mRNA stability in vivo. Our previously published data demonstrate that both proteins are involved in thyroid carcinogenesis. Several other reports postulate that mRNA binding proteins may participate in thyroid hormone actions. However, until now, exacts mechanisms and the possible role of post-transcriptional regulation and especially the role of AUF1 and HuR in those processes remain not fully understood. In this study we shortly review the possible function of both proteins in relation to development and various physiological and pathophysiological processes, including thyroid function and disorders.
... Most of THs response genes are positively (Ishiguro et al., 2010;Calvo and Obregon, 2009), while few are negatively regulated by T3. TSH gene is one of THs down-regulated genes, and its expression has been shown to be controlled by T3 at the transcriptional and posttranscriptional levels (Yusta et al., 1998;Staton and Leedman, 1998), being the latter poorly explored. Usually, the posttranscriptional actions do not depend on THs binding to the nuclear receptors (TRs), are achieved in a short period of time, and occur even in the presence of drugs that block gene transcription, which point out to a non-genomic action of THs (Siegrist-Kaiser et al., 1990). ...
Whereas it is well known that T3 inhibits TSHβ gene transcription, its effects on TSHβ mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSHβ transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSHβ transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSHβ mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSHβ mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSHβ transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level.
... Casein mRNA became unstable at the end of lactation. Thyrotropin bsubunit mRNA with the short poly(A) tail is less stable than that with the long one in murine thyrotrope cells (Staton and Leedman, 1998). In ICR mice used here, lactation reaches a maximum at around 13 days postpartum and then declines rapidly (Jara-Almonte and White, 1972). ...
Using beta- and gamma-casein mRNAs, the relationship between poly(A) tail length and half-life of mRNA is determined in the mouse mammary gland during pregnancy and lactation. beta- and gamma-Casein mRNAs increase before and after parturition, respectively. The poly(A) tail as well as the half-life of casein mRNA becomes longer upon the active casein mRNA synthesis. The poly(A) tail is shortened gradually as lactation progresses. The half-life of mRNA decreases approximately from 20 h at early to 4 h at late lactation. Northern blot analysis reveals that nuclear RNA has the same poly(A) tail length as casein mRNA in the cytoplasm does. Thus, the mammary gland changes the poly(A) tail length of casein mRNA. The poly(A) tail length changes in parallel with the level of poly(A) polymerase (PAP) mRNA during pregnancy and lactation, suggesting that the mammary gland determines the poly(A) tail length of casein mRNA through the change in the PAP gene expression. As the half-life of casein mRNA is related with the degree of polyadenylation, we conclude that the poly(A) tail elongation and shortening is a mechanism in regulating the mRNA decay.
... We tested the hypothesis that changes in TR␣1 mRNA turnover may also be involved in the regulation of TR expression, because studies have shown that T 3 reduces the half-life of other mRNAs, including thyrotropin -subunit mRNA (19,34). Using quantitative RT-PCR, we found that endogenous TR␣1 mRNA as well as Ad-TR␣1 mRNA decreased 50% to 70% within4ho fT 3 treatment. ...
Tri-iodo-L-thyronine (T3) is essential for maintaining normal cardiac contractile function by regulating transcription of numerous T3-responsive genes. Both hormone availability and relative amounts of nuclear thyroid hormone receptor isoforms (TRalpha1, TRbeta1) determine T3 effectiveness. Cultured neonatal rat ventricular myocytes grown in T3-depleted medium expressed predominantly TRalpha1 protein, but within 4 h of T3 treatment, TRbeta1 protein increased significantly, whereas TRalpha1 was decreased by 46 +/- 5%. Using replication-defective adenoviruses to overexpress TRalpha1 in cardiomyocytes, we studied the mechanisms by which T3 mediated the decrease in TRalpha1 protein. Inhibitors of the proteosome pathway resulted in an accumulation of ubiquitylated TRalpha1 in the nucleus and prevented T3-induced degradation of ubiquitylated TRalpha1, suggesting that T3 induced proteosome-mediated degradation of TRalpha1; however, TR ubiquitylation was T3 independent. TRalpha1 transcriptional activity, measured using transient transfection of a thyroid hormone-responsive element (TRE) reporter plasmid, was T3 dose dependent and inversely proportional to nuclear TRalpha1 content, with 10 nM T3 having maximum effect. Quantitative RT-PCR showed that both endogenous and adenovirus-expressed TRalpha1 mRNAs were significantly decreased to 54 +/- 11 and 25 +/- 5%, respectively, within 4 h of T3 treatment. Measurements of TRalpha1 mRNA half-life in actinomycin D-treated cardiomyocytes showed that T3 treatment significantly decreased TRalpha1 mRNA half-life from 4 h to less than 2 h, whereas it had no effect of TRbeta1 mRNA half-life. These data support a role for both the proteosome degradation pathway and altered mRNA stability in T3-induced decrease of nuclear TRalpha1 in the cardiomyocyte and provide novel cellular targets for therapeutic development.
... TtT-97 tumours are propagated in hypothyroid mice and they shrink in response to T4 (Brinkmeier et al., 2001). This tumor can be grown in culture by manually dispersing the cells from the tumour mass and plated in media containing 10% T3-depleted FCS (Staton and Leedman, 1998). The presence of either T3 or dexamethasone rapidly decreased the transcription of both the ␣-subunit and TSH genes (Ross et al., 1987;Shupnik and Ridgway, 1985). ...
The pituitary gland is an important component of the endocrine system, and together with the hypothalamus, exerts considerable influence over the functions of other endocrine glands. The hypothalamus either positively or negatively regulates hormonal productions in the pituitary through its release of various trophic hormones which act on specific cell types in the pituitary to secrete a variety of pituitary hormones that are important for growth and development, metabolism, reproductive and nervous system functions. The pituitary is divided into three sections-the anterior lobe which constitute the majority of the pituitary mass and is composed primarily of five hormone-producing cell types (thyrotropes, lactotropes, corticotropes, somatotropes and gonadotropes) each secreting thyrotropin, prolactin, ACTH, growth hormone and gonadotropins (FSH and LH) respectively. There is also a sixth cell type in the anterior lobe-the non-endocrine, agranular, folliculostellate cells. The intermediate lobe produces melanocyte-stimulating hormone and endorphins, whereas the posterior lobe secretes anti-diuretic hormone (vasopressin) and oxytocin. Representative cell lines of all the six cell types of the anterior pituitary have been established and have provided valuable information on genealogy of the various cell lineages, endocrine feedback control of hormone synthesis and secretions, intrapituitary interactions between the various cell types, as well as the role of specific transcription factors that determine each differentiated cell phenotype. In this review, we will discuss the morphology and function of the cell types that make up the anterior pituitary, and the characteristics of the various functional anterior pituitary cell systems that have been established to be representative of each anterior pituitary cell lineage.
... T 3 has been shown to regulate the stability of several other mRNAs. T 3 decreased poly(A) tail length of the pituitary genes thyrotropin -subunit mRNA, a negatively regulated gene, and growth hormone mRNA, a positively regulated gene, decreasing the stability of both mRNAs (20,37,40). T 3 increased the poly(A) tail length of hepatic NADPH-cytochrome P-450 reductase, but only transiently, inasmuch as cytoplasmic poly(A) tail length decreased 16 h later (36). ...
Triiodothyronine (T3) regulates cardiac contractility in part by regulating the expression of several important cardiac myocyte genes. In the rat, the T3-mediated induction of alpha-myosin heavy chain (MHC) transcription in hypothyroid hearts is rapid, exhibiting zero-order kinetics, whereas the repression of beta-MHC in these same hearts is much slower. To elucidate the mechanism for T3 transcriptional as well as posttranscriptional regulation of both MHC gene isoforms, we used an RT-PCR-based transcription assay and the RNA polymerase II inhibitor actinomycin D in an in vivo model to simultaneously measure specific alpha- and beta-MHC heterogeneous nuclear RNA (hnRNA), mRNA kinetics, and MHC antisense RNA. In vivo actinomycin D treatment blocked alpha-MHC transcription in euthyroid rats by >80% at 2 h and suggested a half-life of alpha-MHC hnRNA of approximately 1 h, whereas actinomycin D inhibited beta-MHC transcription in hypothyroid rats by >75% at 6 h, suggesting a significantly longer hnRNA half-life of approximately 4 h. The effect of actinomycin D on beta-MHC transcription was independent of T3. T3 treatment in hypothyroid animals caused beta-MHC mRNA to decline more rapidly than beta-MHC hnRNA, demonstrating, for the first time, a posttranscriptional mechanism(s). The measured change in beta-MHC mRNA half-life indicates a T3-mediated destabilization of beta-MHC mRNA. To understand the mechanism by which T3 destabilizes beta-MHC mRNA, we measured beta-MHC antisense RNA. beta-MHC antisense RNA is present in euthyroid myocytes, but levels are not significant in hypothyroid myocytes. This differential expression may explain some of the effects of T3 on MHC posttranscriptional regulation.
A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form. J. Exp. Zool. 283:510-521, 1999. (C) 1999 Wiley-Liss, Inc.
A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.
Mammalian brain development is controlled by thyroid hormone through the regulation of target genes. In this study, we describe for the first time that a splicing regulator gene is under thyroid hormone control in the rat brain during the critical period of neuronal differentiation. By differential display, we have identified the mammalian homolog of the Drosophila splicing regulator Suppressor-of-white-apricot (SWAP) as a thyroid hormone-regulated gene in an immortal line of rat neuroblasts, E18 cells. Using Northern blotting and in situ hybridization, we found that expression of SWAP is under thyroid control in the developing rat brain. SWAP gene expression is highest during the first 10 days of life (P0-P10), preferentially in cerebral cortex, cerebellum, subventricular epithelium, piriform cortex, hippocampus, amygdala, and caudate putamen. At later stages (P15-P30) SWAP expression decreases, being detectable only in the cerebellum, hippocampus, and layers II/III of cerebral and piriform cortexes. We found that hypothyroidism causes an abnormal high level of SWAP RNA expression at P5-P15 throughout the brain except the cerebellum. Significantly, thyroid hormone treatment in vivo of hypothyroid animals led to a normalization of SWAP RNA expression. Furthermore, similar hormone treatment caused a decrease in SWAP expression in control rats. By modulating the expression of SWAP and perhaps other splicing regulators thyroid hormone may exert wide regulatory effects on multiple genes. The regulation of SWAP gene defines a novel mechanism of action of thyroid hormone which can be important for its effects in the developing brain.
The effects of 3, 3', 5-triiodothyronine (T3) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in the C100 baby hamster kidney cell line. Cells cultured in Minimal Essential Medium (MEM) were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 70.1% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS). When T3 was added to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM for 48 hours, T3 (10(-6) M) treatment rapidly increased HMG-CoA reductase activity, achieving the control level six hours after treatment. Such effects of T3 were blocked by actinomycin D (5 microg/ml) or cycloheximide (10 microg/ml). The transcriptional rate of the HMG-CoA reductase gene did not change significantly regardless of the presence of T3, while T3 inhibited the 25-hydroxycholesterol-mediated decay of the reductase mRNA significantly. Our results show that T3-dependent regulation of HMG-CoA reductase activity, via the de novo synthesis of the reductase enzyme, seems to be mediated at least partially by the stabilization of HMG-CoA reductase mRNA.
The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.
Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H(2)O(2), nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363-27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.
The regulation of mRNA decay is a major control point in gene expression. The stability of a particular mRNA is controlled by specific interactions between its structural elements and RNA-binding proteins that can be general or mRNA-specific. Regulated mRNA stability is achieved through fluctuations in half-lives in response to developmental or environmental stimuli like nutrient levels, cytokines, hormones and temperature shifts as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury. Furthermore, in specific disorders like some forms of neoplasia, thalassemia and Alzheimer's disease, deregulated mRNA stability can lead to the aberrant accumulation of mRNAs and the proteins they encode. This review presents a discussion of some recently identified examples of regulated and deregulated mRNA stability in order to illustrate the diversity of genes regulated by alterations in the degradation rates of their mRNAs.
The tau gene encodes a microtubule-associated protein expressed by neuronal and glial cells. Abnormal deposits of Tau protein are characteristic of several neurodegenerative disorders. Additionally, mutations affecting tau pre-mRNA alternative splicing of exon 10 are associated with frontotemporal dementia and Parkinsonism linked to chromosome 17. In rodents, this process is developmentally regulated by thyroid hormone (T3) causing the predominance of exon 10-containing transcripts. Here we demonstrate that musashi-1 (msi-1) gene is induced by T3 during rat brain development and in N2a cells. T3 increases msi-1 mRNA level in an actinomycin D-sensitive, cycloheximide-resistant fashion without affecting its half-life, which suggests a transcriptional effect. Both ectopic Msi-1 expression and T3 treatment increased the proportion of exon 10-containing tau transcripts. Furthermore, antisense msi-1 expression inhibited T3 action. Our results show that msi-1 mediates the posttranscriptional regulation of tau expression by T3.
Regulation of gene expression is essential for the homeostasis of an organism, playing a pivotal role in cellular proliferation, differentiation, and response to specific stimuli. Multiple studies over the last two decades have demonstrated that the modulation of mRNA stability plays an important role in regulating gene expression. The stability of a given mRNA transcript is determined by the presence of sequences within an mRNA known as cis-elements, which can be bound by trans-acting RNA-binding proteins to inhibit or enhance mRNA decay. These cis-trans interactions are subject to a control by a wide variety of factors including hypoxia, hormones, and cytokines. In this review, we describe mRNA biosynthesis and degradation, and detail the cis-elements and RNA-binding proteins known to affect mRNA turnover. We present recent examples in which dysregulation of mRNA stability has been associated with human diseases including cancer, inflammatory disease, and Alzheimer's disease.
Adrenomedullin, a vasodilatory peptide originally isolated from pheochromocytoma, is known to regulate cell growth, apoptosis, and migration. Overexpression of the c-myc oncogene has been shown to suppress the mouse adrenomedullin gene via the initiator element. We investigated whether c-myc regulates rat and human adrenomedullin genes because there appears to be no initiator elements in their promoter regions. Transactivation of the human adrenomedullin gene by c-myc was demonstrated using a luciferase reporter construct containing an upstream sequence. Using a panel of isogenic rat fibroblast cell lines with differential c-myc expression obtained by targeted homologous recombination, we found markedly elevated adrenomedullin transcript levels in cells stably overexpressing c-myc but a minimal decrease in two independent cell lines containing a homozygous null deletion of c-myc. Degradation of adrenomedullin mRNA was enhanced by a c-myc transgene, resulting in a relatively reserved increase in cellular secretion of adrenomedullin-like immunoreactivity. These results indicate that c-myc transactivates rat and human adrenomedullin genes and accelerates the degradation rate of adrenomedullin mRNA. However, c-myc is not essential for basal expression of the adrenomedullin gene.
Thyrotropin (thyroid stimulating hormone, TSH) is a member of the pituitary glycoprotein hormones, consisting of two dissimilar subunits, alpha and beta. The two subunits are produced by different genes and are regulated independently. We have previously cloned a TSHbeta cDNA from bighead carp pituitary and investigated its gene regulation. We report here the direct effects of mammalian TSH-releasing hormone (TRH), leptin, neuropeptide-Y (NPY), beta-endorphin and galanin on mRNA levels of both TSHbeta and alpha-subunits in the pituitary of bighead carp in vitro. The dispersed pituitary cells of bighead carp were incubated at 25 degrees C for 6 h with different doses of these factors. The relative mRNA levels of TSHbeta and alpha-subunits were estimated by traditional polymerase chain reaction (PCR) analysis and fluorescence real-time PCR analysis. The results revealed that mammalian TRH, leptin and beta-endorphin produced dose-dependent stimulatory effects on mRNA levels of both TSHbeta and alpha-subunits while thyroxine (T4) and mammalian galanin suppressed mRNA levels of both TSHbeta and alpha-subunits. NPY suppressed TSHbeta mRNA level, but stimulated alpha-subunit mRNA level. This study has demonstrated that mammalian TRH, leptin, NPY, beta-endorphin and galanin were active in modulating the steady-state mRNA levels of TSHbeta and alpha-subunits of bighead carp pituitary in vitro. The results suggest that endogenous TRH, leptin, NPY, beta-endorphin and galanin may modulate transcript levels of TSHbeta and alpha-subunits in pituitary of bighead carp.
A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.
Whereas the role of thyroid hormone is clearly established in the regulation of cholesterol homeostasis, its involvement in the control of serum triglyceride (TG) levels remains largely debated. Angiopoietin-like proteins 3 and 4 have recently been characterized as potent lipoprotein lipase inhibitors and therefore as important components of plasma triglyceride homeostasis. In the present study, the role of thyroid hormone in the regulation of both ANGPTL4 and ANGPTL3 gene expression was investigated. In vivo studies revealed that thyroid hormone down-regulates ANGPTL3 but not ANGPTL4 gene expression in hypothyroid rats. Using thyroid hormone receptor (TR)-deficient mice, we show that thyroid hormone regulates ANGPTL3 gene expression in a TRbeta-dependent manner. Transfection studies revealed that this inhibition occurs at the transcriptional level in a DNA binding-independent fashion and requires the proximal (-171 to +66) region of the ANGPTL3 gene promoter. Moreover, site-directed mutagenesis experiments indicate that the HNF1 site within this proximal region mediates this TRbeta-dependent repression. Finally, co-transfection studies and electrophoretic mobility shift assays suggest that TRbeta antagonizes the HNF1alpha signaling pathway by inhibiting its transcriptional activity without interfering with its DNA-binding capacity. Taken together, our results lead to the identification of ANGPTL3 as a novel TRbeta target gene and provide a new potential mechanism to explain the hypotriglyceridemic properties of TRbeta agonists in vivo.
For better understanding of phylogenetic diversity and evolution of pituitary thyroid-stimulating hormone (TSH) in birds, we have cloned the cDNAs encoding TSH beta subunit (TSHbeta), by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) from two species of domestic ducks, common duck (Tsaiya duck and Pekin duck) (Anas platyrhynchos domesticus) and mule duck (hybrid of male muscovy duck Cairina moschata and female A. platyrhynchos domesticus). The nucleotide sequences of isolated TSHbeta cDNAs of the two species of ducks are identical, with each including 66 bp of 5'-untranslated region (UTR), 402 bp of coding region, and 82 bp 3'-UTR followed by 18 bp poly A tract. The deduced TSHbeta subunit of the ducks contains 134 amino acids consisting of a putative signal peptide of 19 amino acids and a putative mature protein of 115 amino acids. However, the TSHbetas of common duck and mule duck differ from the TSHbeta of muscovy duck in one amino acid at position 97 of the mature protein: isoleucine for common duck and mule duck, and valine for muscovy duck. Our findings thus demonstrate that inter-genus variation of TSHbeta exists in Family Anatidae, and that TSHbeta gene in the mule duck is preferentially transcribed from the maternal genome rather than from the paternal genome. TSHbeta mRNA expressions were investigated by culturing common duck pituitaries with various doses of hormones. Thyrotropin-releasing hormone (TRH) stimulated, while thyroid hormones, triiodothyronine (T(3)) and thyroxine (T(4)), inhibited the TSHbeta mRNA levels, in dose-related manners. The findings thus support that the mode of regulation of TSH gene expression in hypothalamo-pituitary-thyroid axis in birds is similar to that in mammals. Cortisol and corticosterone decreased the steady-state TSHbeta mRNA levels at the pituitary level, in a dose-related manner, the first-time demonstration in vertebrates. The results may suggest that glucocorticoids exert suppressive action directly at pituitary level in modulation of steady-state TSHbeta mRNA level.
The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXRbeta and RXRgamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXRbeta and RXRgamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXRbeta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXRgamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXRbeta gene was positively regulated by TH, whereas the transcription of RXRgamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXRbeta is positively regulated by TH at transcriptional level, while the negative regulation of the RXRgamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXRbeta and RXRgamma.
GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals.
In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.
The mechanisms by which c-fos mRNA is targeted for decay have been examined. Rapid removal of the poly(A) tail occurs before the transcribed portion of the c-fos message is degraded. Identification of the determinants that mediate c-fos message deadenylation reveals that they coincide directly with previously characterized determinants of c-fos mRNA instability, one in the protein-coding region and the other an AU-rich element (ARE) in the 3'-untranslated region. Insertion of either of these c-fos instability elements into the stable beta-globin message confers the property of rapid deadenylation. Mutation of the ARE indicates that this sequence controls two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require intact AUUUA pentanucleotides within the ARE, and subsequent degradation of the transcribed portion of the message, which appears to be dependent on the AUUUA pentanucleotides. These results indicate that structurally distinct instability determinants within the transcribed portion of labile messages can function by promoting rapid removal of the poly(A) tail as a first step in the decay process.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.
Post-transcriptional regulation of transferrin receptor mRNA levels by iron is mediated by a portion of the 3' untranslated region (UTR) of the mRNA. We have previously shown that a 678 nucleotide fragment of the 3'UTR contains the regulatory element(s). Within this region are five RNA structures which resemble the iron-responsive element (IRE) in the 5' untranslated region of the ferritin mRNA which is regulated translationally by iron. The IREs from the ferritin and transferrin receptor mRNAs compete in an in vitro assay for interaction with a cytoplasmic protein; the activity of this IRE-binding protein is dependent upon the iron status of the cells. Based on further deletion analysis reported here, the sequence required for iron regulation of the transferrin receptor have been limited to 250 nucleotides which we have produced synthetically and cloned. This sequence, which contains three IREs, is capable of producing iron-dependent regulation of transferrin receptor levels. Removal of the three IREs from the synthetic element results in loss of iron regulation. Moreover, deletion of a single cytosine residue from each of the three IREs in the synthetic regulatory element eliminates high-affinity binding to the IRE-binding protein in vitro and results in low levels of iron-independent transferrin receptor expression, consistent with production of a constitutively unstable mRNA. These data indicate that the ability of the mRNA to interact with the IRE-binding protein is required for regulation of transferrin receptor mRNA levels by iron.(ABSTRACT TRUNCATED AT 250 WORDS)
The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide
containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved
among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal
domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site
with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple
N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.
Thyrotropin (TSH), a pituitary glycoprotein, consists of two subunits (alpha and beta), and regulates thyroid hormone production by the thyroid gland. The thyroid hormones, in turn, feedback negatively on TSH production. We have investigated the effect of triiodothyronine (T3) on the transcription of the TSH subunit genes. Hypothyroid LAF1 mice bearing TtT 97 thyrotropic tumors were injected daily with T3 (5 micrograms/100 g of body weight, intraperitoneal) and killed after 0, 0.5, 1, 4, or 120 h. Nuclei were isolated from the tumors and allowed to continue RNA synthesis in the presence of [alpha-32P]UTP. Newly synthesized mRNA sequences were quantitated by hybridization to immobilized cloned cDNA containing sequences specific for either alpha subunit or TSH-beta mRNA. Basal levels of mRNA synthesis were similar for alpha subunit and TSH-beta. After only 30 min of T3 treatment in vivo, mRNA synthesis was decreased for both alpha subunit (by 28%) and TSH-beta (by 61%). These decreases were maximum after 4 h, (75% for alpha subunit, greater than 95% for TSH-beta mRNA synthesis), and were maintained for the 5 days of T3 treatment. Decreases in mRNA synthesis precede decreases in tissue mRNA concentrations. Our studies suggest that T3 rapidly decreases the transcription of both the alpha subunit and TSH-beta genes, and that the transcription of the TSH-beta gene is affected to a greater extent than the alpha subunit gene.
We have studied the regulation of the biosynthesis of thyrotropin (TSH) and its alpha and beta subunits by thyroid hormone in thyrotropic tumors carried in hypothyroid mice. Treatment with 3,5,3'-triiodo-L-thyronine (T3) (20 micrograms/100 g, body weight) daily for 4 or 10 days reduced serum TSH to 3 and 0.3% of control, respectively. Serum levels of free alpha subunit were reduced to 60 and 11% of control at 4 days and 10 days, respectively, and serum free TSH-beta was undetectable at both time points. There was no significant decrease in tumor TSH content after 4 days of treatment and, after 10 days, TSH content was reduced to 15% of control levels. There was no significant effect of T3 on tumor alpha subunit levels at either 4 or 10 days. In contrast, tumor TSH-beta content was markedly reduced after 4 days and 10 days of T3 treatment, to 29 and 10% of control levels, respectively. Translation of tumor poly(A) mRNA in a rabbit reticulocyte lysate system showed that thyroid hormone decreased translatable TSH-beta mRNA to undetectable levels at both 4 and 10 days, whereas translatable alpha mRNA was reduced strikingly only at 10 days in one of two tumors. RNA blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs showed that TSH-beta mRNA was reduced to less than 10% of control after both 4 and 10 days of T3 treatment, whereas, again, alpha mRNA was only reduced in one of two tumors at 10 days. Our data thus show that thyroid hormone affects alpha and TSH-beta mRNA and protein levels discordantly and suggest that regulation of TSH biosynthesis may occur predominantly at the level of TSH-beta mRNA.
This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end.
AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.
The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXR beta and RXR gamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXR beta and RXR gamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXR beta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXR gamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXR beta gene was positively regulated by TH, whereas the transcription of RXR gamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXR beta is positively regulated by TH at transcriptional level, while the negative regulation of the RXR gamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXR beta and RXR gamma.
The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T3 augmented an iron-induced increase in CAT activity by approximately 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T3-receptors may be necessary for this T3-induced response, because the effect could not be reproduced by the addition of T3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T3-receptors. We conclude that T3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.
We studied the regulation of mRNAs encoding the α- and β-subunits of TSH by thyroid hormones (T4 and T3) in mouse thyrotropic tumors and pituitary glands. Hypothyroid male (LAF1) mice bearing thyrotropic tumor (TtT97) were injected daily with T4 for 0, 1, 5, 12, or 33 days. After day 33, plasma levels of TSH and free (unassociated) TSH β-subunit were reduced to less than 1% of control levels, whereas free α-subunit was reduced to 6% of control levels. Steady state levels of subunit mRNAs in extracts of the thyrotropic tissues were measured by blot hybridization analyses using mouse subunit-specific cloned cDNAs. Treatment of mice with T4 caused a rapid decline in the levels of tumor mRNAs for both α and TSHβ; after day 1, α and TSHβ mRNA levels decreased to 35% and 10% of control values, respectively. Levels of TSHβ mRNA were undetectable after 5 days of T4 treatment, whereas levels of α-subunit mRNA remained at 30-35% of control levels even after day 33. In a separate experiment, TSHβ mRNA decreased to 42% of the control level (P < 0.05), whereas α-subunit mRNA remained at 64% of the control level (P = NS) 4 h after a single injection of T4. Finally, T3 also caused a rapid decrease in the levels of both subunit mRNAs in the anterior pituitary glands of hypothyroid mice, but the effect was more complete on TSHβ mRNA levels. We conclude that thyroid hormones have rapid suppressive effects on the levels of mRNAs encoding the subunits of mouse TSH in the thyrotrope. The suppressive effects of thyroid hormones occur more rapidly and are greater for TSHβ than α-subunit mRNAs. The parallel changes observed in the subunit mRNA levels and the plasma subunit protein levels in animals treated with thyroid hormones suggest that the changes in the plasma levels of TSH and subunits may reflect effects of thyroid hormones on TSH gene expression in addition to effects on secretion.
The regulation of mRNA stability is an important step in the control of gene expression. Characterization of the mechanisms involved in the turnover of individual mRNAs has identified a requirement for specific cis-acting sequences and trans-acting factors, as well as an involvement of the translation apparatus. In the past year, significant progress has been made in the identification of trans-acting factors by both biochemical and genetic approaches. This review summarizes that progress and promotes the notion that the ribosome itself should also be considered as a trans-acting component of the mRNA decay machinery.
To determine whether thyrotropin (TSH) suppression was characterized by a rapid and slow component in euthyroid rats, TSH concentrations were measured in concentrates of plasma pooled from groups of 8 euthyroid rats 24 hr after injection of a single dose of triiodothyronine (T3). Plasma TSH decreased after T3 injection to values 3.2-5.9% of pre-T3-injection concentrations. The presence of TSH in plasma after single T3 injection and its absence (<0.25% of euthyroid values) after euthyroid rats were treated with T3, 2-4 μg/day for 43 days, indicate that, as in the hypothyroid rat, TSH suppression has both a rapid and slow component in the euthyroid rat. A small but statistically insignificant increase in TSH secretion occurred after injection of a high dose of thyrotropin-releasing hormone (TRH; 1 μg/100 g BW) into T3-treated euthyroid or hypothyroid rats. This suggested that endogenous TRH did not play an important role in maintaining TSH secretion in the presence of high plasma T3 concentrations. The metabolic clearance rate (MCR) of TSH was measured to determine whether altered rates of TSH metabolism could account for the 30-100-fold greater TSH concentration observed after 24 hr in T3-injected hypothyroid rats as compared to T3-injected euthyroid rats. Neither a mean 40% decrease in the MCR of TSH nor a 4-fold increase in number of thyrotrophs in hypothyroid rats could account for the large difference in residual TSH concentration. The TSH secretion rate per thyrotroph in non-injected or T3-injected hypothyroid rats appears to be 4-8-fold greater than in euthyroid rats.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
Primary suspension cultures from serially transplanted mouse pituitary thyrotropic tumors were shown to be regulated by physiological levels of thyroid hormones. TSH release was linear for up to 48 h in control cultures and was inhibited progressively after 10, 24, and 48 h in cultures exposed to triiodothyronine (T3), 4.0 nM. TSH release was inhibited up to 55% of control and glucose consumption was stimulated up to 2.6-fold in a dose-dependent fashion by T3 between 0.2 and 4.0 nM. A biphasic dose-dependent relationship for T3 and thyroxine (T4), and TSH production was demonstrated in two series of cultures. TSH production was stimulated progressively by T3 up to 0.1 nM and T4 up to 5 nM. At higher T3 and T4 levels, TSH production was progressively inhibited. Half-maximal inhibition occurred at total medium concentrations of 0.2 nM T3 and 15 nM T4 or free hormone levels of 8 x 10-12 M and 14 x 10-11 M, respectively. High affinity, low capacity nuclear binding sites were demonstrated for T3 and T4. The apparent equilibrium dissociation constants were 0.16 nM for T3 and 1.7 nM for T4 in serum-free medium. There were approximately 14,000 molecules of T3 or T4 bound per cell nucleus at saturation. These data suggest that T3 is approximately 15-fold more potent than T4 in regulating TSH production and cellular metabolism in these cells, and that these effects may be initiated by interaction with nuclear binding sites. Also, that within the physiological range, low levels of thyroid hormones stimulate and higher levels inhibit TSH production.
A method combining enzymatic cellular dispersal, direct cell counting, differential cell counts at the electron microscope level and DNA determinations was devised and employed for determination of the cell numbers of each anterior pituitary cell type in euthyroid (E) and hypothyroid Tx) rats. Pituitaries from Tx rats had increased cell number as demonstrated by a mean 33.7% increase in DNA content (microgram DNA/pituitary). Total cells increased from (3.14 +/- 0.36) X 10(6) in E rats to (3.98 +/- 0.27) X 10(6) in Tx rats. P less than 0.005. The cellular DNA content (g/cell) in E rats ,10.84 +/- 0.63 (SD), was indistinguishable statistically from that of Tx rats, 11.24 +/- 0.52. Cell distribution among various pituitary cell types was virtually identical when determined in pellets from dispersed cells and randomized solid tissue from the same groups of E and Tx rats. These data indicated that there was no selective cell loss during the cell dispersion procedure. Major changes in Tx rats compared to E rats were a marked increase in percentage of thyrotrophs, from 10.7 +/- 1.75 (E) to 34.4 +/- 1.0 (Tx), and a decrease in percentage of somatotrophs, from 55.3 +/- 1.82 to 15.3 +/- 0.97. The calculated cell distribution showed that the number of thyrotrophs increased from 0.34 +/- 0.02 to 1.37 +/- 0.05 millions per pituitary and somatotrophs decreased from 1.74 +/- 0.11 to 0.61 +/- 0.02 millions in hypothyroid rats. The method described herein thus provides a quantitative estimate of changes in pituitary cell populations in different hormonal states and should be useful in studies of the kinetics of pituitary cell replication and removal.
In thyroid hormone-depleted rats, the rate of transcription of the growth hormone (GH) gene in the anterior pituitary gland
is lower than the rate in euthyroid controls, and there is a corresponding reduction in the abundance of the GH mRNA. Concomitantly,
the poly(A) tail of the GH mRNA increases in length. Examination of nuclear RNA from anterior pituitary glands of control
and thyroid hormone-depleted rats revealed no difference in the length of pre-mRNAs containing the first and last introns
of the GH gene. However, mature nuclear GH RNA is differentially polyadenylated in euthyroid and hypothyroid animals. We suggest
that the extent of polyadenylation of the GH transcript is regulated in the cell nucleus concomitant with or subsequent to
the splicing of the pre-mRNA. Experiments with anterior pituitary gland explant cultures demonstrated that the GH mRNA from
thyroid hormone-depleted rats is more stable than its euthyroid counterpart and that the poly(A) tail may contribute to the
differential stability of free GH ribonucleoproteins.
Thyroid hormone (T3 and T4) down-regulation of TSH subunit steady state mRNA levels and subunit gene transcription in vitro and in vivo has been well studied. We present evidence here that T3 can also regulate the turnover of TSH subunit mRNA. The apparent half-life of the TSH beta-subunit mRNA was determined by adding actinomycin-D (2 microM) to dispersed rat pituitary cultures in hypothyroid medium or medium containing 10(-7) M T3 and analyzing the decline in subunit mRNA levels with time. The half-life of the TSH beta mRNA from those cultures treated with T3 was shorter than that of the control cultures (9 vs. greater than 24 h, respectively). A possible mechanism by which TSH beta-subunit mRNA stability is altered is through a change in the size of each mRNA's poly(A) tail. Northern blot analysis of total RNA from the above cultures revealed that T3 treatment reduces the size of the TSH beta-subunit mRNA. To determine if this alteration of mRNA size was due to a loss of a portion of the poly(A) tract and not to alternative splicing of the transcript or use of a secondary transcriptional start site, pooled RNAs were hybridized with oligo(dT) and subsequently digested with RNAse-H to remove the poly(A) tract. RNA blot analysis of these RNAs showed that T3 treatment results in the loss of most of the TSH beta poly(A) tail.(ABSTRACT TRUNCATED AT 250 WORDS)
Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.
We developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization
after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region.
Using this method, we studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes
in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin
mRNA in hydrated rats was, quite remarkably, approximately 250 nucleotides in length, in contrast to that of somatostatin
mRNA, which was approximately 30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from approximately
250 to approximately 400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its
removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic
mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of
specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation.
A variety of rapidly growing mammalian cells contain a substantial portion of their actin mRNA in a poly(A)- form. We have used DNA-driven hybridization of a cloned actin cDNA-containing plasmid with pulse-labeled RNA from mouse S-180 ascites cells to examine newly synthesized actin mRNA. Our results indicate that the same proportion of newly synthesized and steady-state actin mRNA (approx. 40%) exists in a poly(A)- deficient form. This suggests that the poly(A)- form arises by some process other than slow cytoplasmic de-adenylation of a poly(A)+ precursor. We have also examined cell cycle-enriched populations of S-180 ascites cells for the presence of poly(A)- actin mRNA. Results from these experiments indicate that cells in G1 phase of the cell cycle contain predominantly poly(A)+ actin mRNA, while the poly(A)- form is restricted to late-S and post-S phase cells.
We have investigated the direct effect of the thyroid hormone T3 on the TSH subunit genes in tissue explants. Minces of TtT 97 thyrotropic tumor were treated with 5 nM T3 for varying periods of time. Nuclei were then isolated from the tumor cells and allowed to continue RNA synthesis in the presence of [alpha-32P]UTP. Newly synthesized RNA sequences were quantified by hybridization to immobilized cloned cDNAs containing sequences specific for either TSH beta or alpha-subunit mRNA. Basal TSH beta and alpha-subunit mRNA synthesis rates were both approximately 300-400 parts/million, the same as in vivo values. After 15 min of T3 treatment, TSH beta mRNA synthesis was significantly decreased by 42% and was maximally decreased by 95% after 1 or more hours of T3. Synthesis of alpha-subunit mRNA was decreased by 38% after 30 min of T3 treatment and by 78% after 1 h or more of T3. The suppressive effects of T3 on transcription correlated with the time course of T3 binding to its nuclear receptor. These changes are quantitatively similar to those observed after in vivo T3 treatment. Decreases in mRNA synthesis preceded significant decreases in tissue steady state mRNA levels or subunit protein levels. The presence of the protein synthesis inhibitor cycloheximide (25 micrograms/ml) during a 4-h incubation with T3 did not change the T3-mediated decreases in TSH beta or alpha-subunit mRNA synthesis or the decreases in cellular mRNA levels. Therefore, T3 can act directly on the thyrotrope to suppress TSH beta and alpha-subunit mRNA synthesis, and protein synthesis is not necessary for the T3-mediated decreases in gene transcription. The data suggest that T3 may act directly at the level of the TSH subunit genes to modulate their expression.
Neuronal and glial cell-enriched nuclei were prepared from developing rat brain cortex to study the effect of thyroxine on nuclear poly(A) polymerase. Long-term thyroxine treatment stimulated the activity of the chromatin-associated enzyme of neuronal nuclei without significantly affecting that of glial nuclei. The nuclear content of poly(A)-containing RNA in neuronal nuclei was also increased by thyroxine administrations. A single dose of thyroxine enhanced both the chromatin-bound and the free, nucleoplasmic form of neuronal poly(A) polymerase in hypothyroid rats aged 12 days. The results suggest that thyroid hormones may regulate both transcriptional and post-transcriptional events in target cell nuclei.
We have observed three effects of deletion mutations on polyadenylation of late SV40 mRNAs. The first class of mutants lack segments (-3 to -14 bp) between the 5-AAUAAA-3' and normal poly(A) site. These mutants produce mRNas polyadenylated at new sites, downstream from the wild-type site. The poly(A) site is moved farther downstream as the deletions become larger; as a result, polyadenylation always occurs within an 11-19 nucleotide range from the AAUAAA sequence. The second class of mutants lack segments (-12 to -30 bp) between the AAUAAA sequence and the coding region of the mRNA. The poly(A) site for only one of these mutants was studied (dl1457, -12 bp). In this case, the spatial relationship between AAUAAA and poly(A) site is altered. dl1457 produces a class of mRNAs polyadenylated at the first Ca following the AAUAAA sequence, as well as other mRNAs polyadenylated farther downstream. Finally, a 16 bp deletion that includes the AAUAAA sequence prevents poly(A) addition.
This chapter discusses the regulation and organization of thyroid stimulating hormone (TSH) genes. The chapter discusses the development of highly sensitive and specific radioimmunoassays for each subunit after the dissociation of human TSH and purification of the α and β subunits. In an experiment described in the chapter, subunit secretion and regulation in clinical studies in humans were observed. In addition, extracts of normal human pituitary glands obtained postmortem were prepared, and the extracts were fractionated by gel chromatography on Sephadex G-100. In spite of variability in subunit content among normal pituitaries, a consistent excess of free α subunits relative to the sum of all free pituitary β subunits was demonstrated in each normal pituitary, with an α-to-β subunit ratio ranging from 1.3 to 8.3. These studies supported the concept that biosynthesis of the unique β subunits was limiting in the production of complete glycoprotein hormones.
A small region (220 bases) of SV40 sequence information--141 bases before the polyadenylation site and 79 beyond--are sufficient for cleavage of an messenger RNA precursor (that is, the formation of a mature 3' terminus), the addition of polyadenylic acid, and the transport of messenger RNA from the nucleus to the cytoplasm. These 220 bases include a highly conserved sequence--AAUAAA (A, adenine; U, uracil). Four point mutations in this sequence--AACAAA, AAUUAA, AAUACA, and AAUGAA (C, cytosine; G, guanine)--prevent cleavage.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.
We have previously demonstrated divergent changes in pituitary free TSH beta and alpha-subunit concentrations in hypothyroid mice during prolongation of experimental hypothyroidism and after T4 administration. This report compares the simultaneous responses of pituitary and thyrotropic tumor TSH, TSH beta, and alpha-subunit to 12 days of T4 administration in four groups of hypothyroid LAF1 mice bearing thyrotropic tumors. Half of each group received daily injections of T4 (10 micrograms/100 g BW, ip) for 12 days; the other half of each group received saline. Plasma concentrations of TSH, free TSH beta, and free alpha-subunit were suppressed by T4 administration to 0.1-1.5%, 2.0-3.9%, and 4.1-25% of control concentrations, respectively. Pituitary TSH and free TSH beta concentrations fell significantly with treatment to 24-43% and 10-28% of control concentrations, respectively. In contrast, pituitary alpha-subunit concentrations did not fall (they were 106-203% of control values), and a rise in the pituitary alpha-subunit concentration was statistically significant in one group (P less than 0.02). Thyrotropic tumor TSH and free TSH beta concentrations fell significantly with treatment to 9-31% and 8-35% of control concentrations, respectively. Tumor alpha-subunit concentrations did not fall (they were 87-195% of control values), and a 2-fold rise was statistically significant in one group (P less than 0.05). Both pituitary and tumor alpha-subunit to TSH beta molar ratios increased significantly with T4 administration in all groups. We conclude that (1) pituitary and thyrotropic tumor TSH beta concentrations fall after 12 days of T4 administration while alpha-subunit concentrations are unchanged or actually rise; (2) this divergent response is qualitatively similar in hypothyroid mouse pituitary and thyrotropic tumors; and (3) these data suggest differences between the regulation of intracellular TSH beta and alpha-subunit.
A repeating structure of cytoplasmic poly(A)-ribonucleoprotein is revealed by digestion with T2 RNase. A pattern of fragments that are multiples of about 27 residues is obtained. The repeating structure is readily reconstituted from purified poly(A) and cytoplasmic factors. Reconstitution is specific for poly(A), as shown by the lack of competition by poly(G), poly(C), poly(dA), and tRNA. The repeating structure is absent from the nucleus, and so appears to be formed upon transport to the cytoplasm.
We have developed a dispersed cell monolayer system derived from bovine anterior pituitary glands. Fresh 1- to 6-week-old calf anterior pituitaries were mechanically and enzymatically dispersed and incubated with Dulbecco's Modified Minimal Essential Medium containing 10% hypothyroid goat serum. The media and cell extracts from confluent monolayers were analyzed for bovine TSH and free α, and TSHβ subunits by specific homologous RIAs. Basal levels of TSH, free α, and free TSHβ subunits in the media were 6.2 ± 0.3, 8.0 ± 0.3, and 0.95 ± 0.05 ng/10 6 cells.24 h, respectively. Hence, an 8- to 10-fold excess of free α over free TSHβ subunits was released into the medium. Intracellular basal levels of TSH, free α, and free TSH̄ subunits were 27.6 ± 1.7, 10.7 ± 0.2, and 2.6 ± 0.3 ng/10 6 cells.24 h, respectively, and indicated a 3- to 4-fold excess of free α over free TSHβ subunits within the cells. The total α-subunit to total β-subunit ratio was 2:1. TRH stimulated release of TSH and its subunits in a dose-dependent fashion, with a half-maximal dose of 2 nM and a maximal response dose of 10 nM. Stimulation with 100 nM TRH increased the levels of TSH, free α, and free TSHβ subunits (450-900%, 180-200%, and 300-400%, respectively) in medium, with concomitant decreases within cells. Treatment with thyroid hormones decreased basal and blunted TRH-stimulated levels of TSH and its subunit in medium but had no effect on intracellular stores. However, large doses of T 4 (25 nM) of T 3 (1 nM) did not completely abolish the TRH (100 nM)-stimulated hormone response. TRH and thyroid hormones affect the release of TSH and TSHβ to a greater extent than they do the α-subunit. Finally, total α and TSHβ subunit production was increased with TRH stimulation and decreased with thyroid hormone exposure. Thus, an in vitro system to study the net production and secretion of TSH and its subunits in the normal thyrotrope has been established.
Thyroid hormone (T3) regulates the expression of rat TSH beta-subunit (TSH beta) mRNA, in part, at the posttranscriptional level, by reducing the half-life of TSH beta mRNA. The mechanism(s) mediating this alteration in mRNA stability are unknown, but previous work indicates that labile protein(s) are involved. The majority of cis-acting elements identified to date that have been implicated in the regulated destabilization of mRNAs have been located in the 3'-untranslated region (3'-UTR) of the mRNA. The 3'-UTR of rat, murine, and human TSH beta mRNA is highly conserved, and within this region is a 12-nucleotide consensus sequence, which is shared by the 3'-UTR of several other genes with unstable mRNAs. We reasoned that this homologous region could represent a binding motif for specific trans-acting RNA-binding protein(s), and that identification and characterization of such trans-acting factor(s) may provide critical insight into the mechanisms underlying T3-induced changes in TSH beta mRNA stability. Utilizing the RNA electrophoretic mobility shift assay and analysis of UV cross-linked RNA-protein complexes, a cytoplasmic trans-acting factor of approximately 80-85 kilodaltons was identified from rat pituitaries and several cell lines that binds in a sequence-specific manner to the 3'-UTR of rat TSH beta mRNA. Using competitive antisense oligonucleotides, the predominant binding site was mapped to the first 41 nucleotides of the 3'-UTR, which includes the consensus region. However, sequence upstream of the consensus was also shown to be important for binding. Using RNA electrophoretic mobility shift assay, two mRNAs containing sequence homology with the consensus region, c-erbA alpha-2 and a rat ferritin pseudogene, were shown to specifically compete with rat TSH beta mRNA for binding of this factor. Remarkably, the binding activity of this factor was regulated positively by T3 within 4 h, but only with rat pituitary extracts. These data suggest that in addition to binding rat TSH beta mRNA in a sequence-specific and T3-regulated manner, this novel trans-acting RNA-binding protein may also bind to other cytoplasmic mRNAs involved in diverse intracellular processes.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
To determine pathways of mRNA turnover in yeast, we have followed the poly(A) tail removal and degradation of a pulse of newly synthesized transcripts from four different genes. Before decay of both stable and unstable mRNAs initiated, there was a temporal lag during which the poly(A) tail was deadenylated to an oligo(A) length. Altering the deadenylation rate of an mRNA led to a corresponding change in the length of this lag. The rate of deadenylation and the stability of the oligo(A) species varied between mRNAs, explaining the differences in mRNA half-lives. To examine how the transcript body was degraded following deadenylation, we used the strategy of inserting strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the 3' UTR of mRNAs. Fragments lacking the 5' portion of two different mRNAs accumulated after deadenylation as full-length mRNA levels decreased. Therefore, these results define an mRNA decay pathway in which deadenylation leads to either internal cleavage or decapping followed by 5'-->3' exonucleolytic degradation of the mRNA.
Thyroid hormones suppress the synthesis and release of thyrotropin from thyrotropes in the anterior pituitary gland, a feature that is critical in the classic negative-feedback loop of the pituitary-thyroid endocrine axis. The major effect of thyroid hormones in this system is exerted at the transcriptional level. The molecular mechanisms by which there is negative regulation of TSH subunit gene expression by thyroid hormone have been elucidated. The TSH subunit genes have isolated and characterized. Structure-function analyses using fusion genes and DNA transfection approaches have defined the putative negative TREs among the promoters of the rat, mouse, and human alpha and TSH beta genes. These sequences are either largely overlapping direct TRE half-sites, TRE half-sites as direct repeats gapped by two nucleotides, or single TRE half-sites. These arrangements are distinct from those seen in positive TREs. Recent knowledge regarding the molecular mechanisms of thyroid action in general forces consideration of multiple TR isoforms, TR heterodimer partners (TRAPs), and thyroid hormones in the ultimate mechanisms of negative action. Several models have been proposed, but none has yet been proved. In addition, the role of thyroid hormone in the regulation of gene expression at the posttranscriptional level is beginning to be addressed. Future work should continue to illuminate these important facets of gene regulation.
In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5'-to-3' exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5' and 3' ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.
Based on the above mechanisms of mRNA degradation, an integrated model of mRNA turnover can be proposed (Figure 1). In this model, all polyadenylated mRNAs would be degraded by the deadenylation-dependent pathway at some rate. In addition to this default pathway, another layer of complexity would come from degradation mechanisms specific to individual mRNAs or to classes of mRNAs. Such mRNA-specific mechanisms would include sequence-specific endonuclease cleavage and deadenylation-independent decapping. Thus, the overall decay rate of an individual transcript will be a function of its susceptibility to these turnover pathways. In addition, cis-acting sequences that specify mRNA decay rate, as well as regulatory inputs that control mRNA turnover, are likely to affect all the steps of these decay pathways. One important goal in future work will be to identify the gene products that are responsible for the nucleolytic events in these pathways and to delineate how specific mRNA features act to affect the function of these degradative activities. The identification of distinct mRNA decay pathways should allow, genetic and biochemical approaches that can be designed to identify these gene products. A second important goal is to understand the nature of the interaction between the 5' and 3' termini, which may also be critical for efficient translation.
WolfO1984Theregulationandorganisationofthyroid stimulating hormone genes
- Kouridesia
- Gurrja
KouridesIA,GurrJA,WolfO1984Theregulationandorganisationofthyroid stimulating hormone genes. Recent Prog Horm Res 40:79–11
- Ma Shupnik
- Ec Ridgeway
- Ww Chin
Shupnik MA, Ridgeway EC, Chin WW 1989 Molecular biology of thyrotropin. Endocr Rev 10:459 – 475