Pretranslational Events Research Articles

Cleaved PGAM5 dephosphorylates nuclear serine/arginine-rich proteins during mitophagy

PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.

Transcriptional and post-translational regulatory system for hypoxia specific gene expression using the erythropoietin enhancer and the oxygen-dependent degradation domain

Gene therapy with angiogenic factors is a promising strategy for the treatment of ischemic diseases. However, unregulated expression of an angiogenic factor may induce pathological angiogenesis. In this study, a hypoxia specific gene expression plasmid, pSV-Luc-ODD, was constructed with the oxygen-dependent degradation (ODD) domain for rapid degradation of a target protein under normoxia. In the transfection assay, luciferase activity in the pSV-Luc-ODD transfected cells was much lower under normoxia than that under hypoxia. However, the luciferase mRNA levels under hypoxia and normoxia were not significantly different. Therefore, decrease of luciferase activity under normoxia is not due to pre-translational events such as change of transcription rate or mRNA stability, but to post-translational degradation. For more hypoxia specific gene expression, pEpo-SV-Luc-ODD was constructed with the erythropoietin (Epo) enhancer and the ODD domain. pEpo-SV-Luc-ODD showed more than 1000 times increase of gene expression under hypoxia in Neuro2A cells, compared to normoxia. In addition, reoxygenation studies after hypoxia incubation showed that gene expression was decreased in response to increased oxygen concentration. This highly hypoxia specific gene expression system will be useful for development of targeting gene therapy for ischemic diseases.

Pretranslational Regulation of Type 2 Deiodinase

Type 2 deiodinase (D2) is a highly potent selenoenzyme that catalyzes the first step of thyroid hormone action and its expression is under close, multilevel control. D2 expression is tissue specific and can be regulated by transcriptional and posttranscriptional mechanisms. There is now compelling evidence that D2 is critically important in triiodothyronine (T3) homeostasis as well as in several biologic processes in which thyroid hormone is involved. A host of factors are known to influence dio2 mRNA including the transcription factors that regulate the dio2 gene in different cell types. In particular, cyclic adenosine monophosphate (cAMP) and two homeodomain- containing proteins are relevant for its defined and highly dynamic expression pattern. Single nucleotide polymorphisms of the dio2 gene and other pre-translational events such as alternative splicing can potentially modulate D2 expression and ultimately T(3) generation. Given the wealth of data accumulated over the past two decades, we have attempted in this review to provide a comprehensive update on the pretranslational regulation of D2.

Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.

Effects of Diabetes and Insulin on Hepatic Δ6 Desaturase Gene Expression

It has been recognized that rat liver microsomal Δ6 desaturation activity is defective in experimental diabetes, a fact that may be reverted by means of insulin treatment. In the present study, we used streptozotocin-induced diabetic rats in order to determine the regulatory role of insulin on the expression of hepatic Δ6 desaturase gene. The abundance of hepatic Δ6 desaturase mRNA in the diabetic rats is sevenfold lower than in the control. Insulin administration to diabetic rats induces Δ6 desaturase mRNA eightfold within 24 h. The effect of insulin on the Δ6 desaturase mRNA was inhibited 70% with dibutyryl-cAMP and theophylline administration and 90% by cycloheximide administration. Therefore, our data demonstrate that the activity of hepatic Δ6 desaturase in response to insulin is, at least in part, regulated by pretranslational events that require the synthesis of an unknown protein(s).

UVR modulates the steady-state levels of skin collagen transcripts in hairless mice.

Exposure to solar UV radiation (UVR) leads to changes in the extracellular matrix of the dermis, which is largely composed of collagen and elastin. Collagen and elastin proteins and their corresponding mRNA were assessed in dorsal skins of hairless mice exposed to 0.64 J/cm2 UVR (295-400 nm), 5 days per week, over a 12 week period. A 48% increase in skin-fold thickness was accompanied by increased elastic tissue deposition and more compressed collagen bundles as assessed histologically. Collagen I mRNA levels were similar to those in control skins at 1, 2, 3 and 6 weeks of UVR and less than control levels at 9 and 12 weeks. Collagen III mRNA levels were elevated after 1 week of UVR, remained elevated for a further 2 weeks and then returned to control levels at weeks 6, 9 and 12 when changes are occurring in collagen I transcripts. There was no evidence of corresponding changes in collagen I and III protein levels assessed using electrophoretic techniques. These results suggest that damage to the extracellular matrix, consequent on UVR, is associated with some pretranslational events. Elastin mRNA levels were unaffected by UVR, suggesting that elastic tissue hyperplasia is a posttranscriptional phenomenon.

Cytochrome P4501A1 and cytochrome P4501A2 are downregulated at both transcriptional and post-transcriptional levels by conditions resulting in interferon-alpha/beta induction.

The interferon mediated downregulation of constitutive and inducible cytochrome P450 enzymes occurs through a pretranslational mechanism which depresses the mRNA encoding cytochrome P450s. We measured the transcription rates of CYP1A genes and the turnover of CYP1A mRNA in rats treated with the interferon-alpha/beta inducer polyinosinic acid-polycytidylic acid. The rate of transcription of CYP1A1 and CYP1A2 genes was significantly decreased in hepatic nuclei isolated from male rats treated with polyinosinic acid-polycytidylic acid (10 mg/kg). In addition the rate of degradation of hepatic CYP1A1 and CYP1A2 mRNA was examined following the inhibition of de novo transcription by actinomycin D (1 mg/kg). Messenger RNA levels were analysed by Northern and slot blotting with a 1.2 kb murine CYP1A1 cDNA probe. Interferon significantly augmented the rate of loss of CYP1A1 and CYP1A2 mRNAs suggesting that post-transcriptional degradation of mRNA contributes to the pre-translational events that cause cytochrome P450 downregulation. These results support the involvement of both transcriptional and post-transcriptional mechanisms in the loss of cytochrome P450s mediated by interferon inducers.

Regulation of muscle glucose transport and GLUT-4 by nerve-derived factors and activity-related processes.

Glucose transport and GLUT-4 were examined in muscles in which activity and nerve-derived factors were eliminated (denervation) and in muscles in which only muscle activity was eliminated but in which nerve-derived factors were maintained [tetrodotoxin (TTX) treatment]. After 3 days of denervation, insulin-stimulated 3-O-methylglucose transport was markedly lowered in perfused rat hindlimb muscles (soleus, plantaris, and red and white gastrocnemius; < or = 35%). GLUT-4 was also decreased by 11-65% in denervated muscles. Blocking muscle activity with TTX superfusion of the sciatic nerve for 3 days reduced the insulin-stimulated glucose transport to the same extent as in the denervated muscles (P > 0.05). However, in soleus, plantaris, and red gastrocnemius muscles, GLUT-4 expression was reduced much less by TTX treatment than by denervation (P < 0.05). GLUT-4 mRNA abundance was decreased in denervated muscles but not in TTX-treated muscles. These results suggest that muscle activity largely regulates the insulin-signaling mechanisms of glucose transport and that nerve-derived trophic factors affect pretranslational events to regulate GLUT-4 expression.

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.

Expression of manganese superoxide dismutase in ovine kidney cortex during development.

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes in mammalian tissue. Previous studies have shown that the activity of MnSOD increases in the rat kidney during development. To define further the developmental change in MnSOD activity and better understand some of the steps involved in the control of MnSOD expression during kidney development, we measured MnSOD messenger RNA and enzyme activity in the ovine kidney cortex during fetal life, in the newborn period, and in adults. MnSOD mRNA and enzyme activity were detected at 0.65 gestation. Hybridization of the Northern blot with a human MnSOD cDNA probe showed evidence of two transcripts of 4.0 and 1.5 kb, respectively. There was a significant increase with age of MnSOD activity and MnSOD mRNA (p < 0.0001). The abundance of each MnSOD transcript significantly increased with age (p < 0.001). In the fetuses, both transcripts increase in parallel; in newborns and adults the 1.5-kb increase was significantly greater than the 4.0-kb increase. Enzyme activity and mRNA were strongly correlated (r = 0.89, p < 0.0001). These data indicate that the expression of MnSOD is developmentally regulated in the ovine kidney cortex. This increase seems to be dependent largely on pretranslational events.

Development of pituitary pro-opiomelanocortin gene and peptide expression: characterization and effect of repeated intermittent maternal isolation.

The dynamics of pro-opiomelanocortin (POMC) biosynthesis in the adult rat are altered by demands imposed on the system, such that acute stress increases in the efficiency of anterior pituitary (AP) posttranslational events, while repeated stress increases pretranslational events. In contrast, the developing animal has a limited adrenocortical response to acute stress during the first 2 weeks of life (stress nonresponsive period). In this study, we investigated how the maturing AP and intermediate lobe (IL) POMC cells respond to repeated demand. Measurements of AP and IL POMC mRNA and POMC peptides were performed using Northern gels and radioimmunoassay, respectively. Plasma ACTH and corticosterone measurements were also performed. Maternal isolation, for 1 h on 3 consecutive days, was used as a repeated stress stimulus. The developing AP and IL exhibit an age-related increase in POMC mRNA and peptide levels. On the other hand, AP and IL do not respond to repeated intermittent maternal isolation during the first 2 weeks of life. However, a significant corticosterone release is seen in the 14 and 21-day-old animals. A change in POMC mRNA level is only detected in the 21-day-old AP where levels decrease. Therefore, an adrenocortical response to repeated intermittent maternal isolation predates the appearance of glucocorticoid inhibition of POMC expression in the 21-day-old animal. We propose that an immature neuronal inhibitory circuit during the 3rd week of life causes a sustained corticosteroid response which may in turn trigger AP-delayed feedback.

Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast.

We have isolated and characterized cDNA clones encoding rat cytoplasmic C1-tetrahydrofolate (H4folate) synthase. In eukaryotes, this enzyme is trifunctional and contains the activities of 10-formyl-H4folate synthetase, 5,10-methenyl-H4folate cyclohydrolase, and 5,10-methylene-H4folate dehydrogenase. The deduced sequence of the 935-amino acid open reading frame contained exact matches to NH2-terminal (15 residues) and internal (residues 436-450) peptide sequences obtained from the purified enzyme. The amino acid sequence derived from the rat cDNA shows extensive homology to analogous proteins from bacterial, yeast, and mammalian sources. We have used the cDNA to determine the steady-state levels of the mRNA in various rat tissues and have found that gene expression is regulated in a tissue-specific manner. Transcript levels are highest in kidney and liver with liver transcripts reduced about 30% relative to those found in kidney. Brain, heart, testis, lung and skeletal muscle display even lower transcript levels; reductions range from 70 to 80% of transcript levels found in kidney. Comparison to the levels of enzyme in these tissues allows us to conclude that pretranslational events predominate in the tissue-specific expression. The rat enzyme has been functionally expressed in Saccharomyces cerevisiae as evidenced by its capacity to complement a chromosomal deletion of ADE3, the yeast gene encoding cytoplasmic C1-H4folate synthase.

Postnatal stimulation of rat surfactant protein A synthesis by dexamethasone

The effects of postnatal dexamethasone treatment in vivo on the synthesis of surfactant protein A (SP-A) were examined at the protein and RNA levels. Rats ranging from 1 day old to adult were injected with 200 micrograms of dexamethasone/kg body wt or with vehicle alone and were killed 24 h after injection. One portion of the lung was metabolically labeled with [35S]methionine, the proteins immunoprecipitated using an antiserum to SP-A, and analyzed electrophoretically. Both newly synthesized intracellular and secreted SP-A levels were increased by dexamethasone, reaching averages of 2.3 and 4.5 times control values, respectively. Another portion of the lung tissue was used for RNA analysis. SP-A mRNA levels were also elevated an average of 1.4 times control values by hormone treatment. Dose-response experiments using 16-day-old pups showed that both total SP-A, as measured by enzyme-linked immunosorbent assay, and total SP-A mRNA levels were elevated with dexamethasone treatment, reaching maximal stimulation at 2 mg. We conclude that postnatal dexamethasone treatment in vivo results in increased levels of both newly synthesized SP-A and SP-A mRNA, suggesting that pretranslational events may in part contribute to this process.

Inhibition of gastrin gene expression by somatostatin.

Previous studies performed in this laboratory have demonstrated somatostatin-containing cells in close proximity to gastrin cells in antral mucosa and have shown that somatostatin exerts a local regulatory effect on gastrin release. The present studies were directed to determine whether the effects of somatostatin on the antral gastrin cell involve pretranslational events. The effects of somatostatin on gastrin mRNA were determined by dot blot hybridization using a gastrin antisense RNA probe derived from human gastrin cDNA. Inclusion of somatostatin in the incubation medium caused a dose-dependent inhibition of steady-state gastrin mRNA. Conversely, when antral somatostatin was neutralized by the addition of specific somatostatin antibodies to the incubation medium, gastrin mRNA levels increased by 116 +/- 31% over control values (P less than 0.01). Northern blot hybridization of total antral RNA demonstrated a single major band with a molecular size of approximately 620 nucleotides, closely matching the predicted size of gastrin mRNA. The effect of somatostatin on the rate of gastrin gene transcription was examined using nuclear run-off transcription assays. Inclusion of antibodies to somatostatin in the incubation medium resulted in a 33.8 +/- 3.3% increase in gastrin gene transcriptional activity (P less than 0.01). These studies indicate that, in addition to its established effect on peptide release, somatostatin exerts inhibitory effects on antral gastrin cells at the pretranslational level. Although this inhibition appears to occur in part at the gene transcriptional level, the results also indicate that somatostatin may affect posttranscriptional processing of gastrin mRNA.

Demonstration of thyrotrophin beta-subunit messenger RNA in rat pituitary cells in primary culture--evidence for regulation by thyrotrophin-releasing hormone and forskolin.

We have reported previously the effect of thyroid status in vivo on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding the thyrotrophin (TSH) beta-subunit (Franklyn, Lynam, Docherty et al, 1985). Studies in vitro of the regulation of TSH beta gene transcription have been confined to thyrotrophic tumour cells. We now report the demonstration of TSH beta-subunit mRNA in non-tumorous rat pituitary cells in primary culture. Treatment of cells with thyrotrophin-releasing hormone (TRH) and with forskolin resulted in a marked increase in cellular concentration of TSH beta-mRNA. These results suggest that TRH exerts a direct effect on the pretranslational events involved in TSH synthesis and further that the adenylate cyclase system may be involved in the regulation of synthesis. We have thus described a novel system for the study of TSH beta-subunit gene expression in normal rat pituitary cells in vitro.

Effect of hypothyroidism on pituitary cytoplasmic concentrations of messenger RNA encoding thyrotrophin β and α subunits, prolactin and growth hormone

Thyroid hormones may directly regulate gene expression in the anterior pituitary. In order to examine this possibility we have studied the effect of hypothyroidism in the rat on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding thyrotrophin (TSH) beta and alpha subunits, prolactin and GH. We demonstrated a marked increase in TSH beta and alpha subunit mRNA, accompanied by a decrease in GH mRNA, in the hypothyroid state, changes largely reversed by thyroid hormone replacement. We have thus shown a direct influence of thyroid status on the pretranslational events occurring in pituitary hormone synthesis. The simultaneous rise in cytoplasmic TSH beta and alpha mRNA levels and fall in GH mRNA in hypothyroidism suggests that thyroid status exerts a differential effect on the expression of these genes.

Pretranslational regulation of ectopic hCGα production in ChaGo lung cancer cells by sodium butyrate

Ectopic production of hCG and its free α subunit by ChaGo lung cancer cells is stimulated by sodium butyrate. To investigate pretranslational regulation in this system, we examined the response of the hCGα and β subunit mRNAs in ChaGo-Kl cells, a clone that produces free hCGα but no hCG or hCGβ in the basal state. When a Northern blot of total RNA from ChaGo cells was hybridized to a [ 32P]- labeled hCGα cDNA probe, a single band was detected that was identical in size (∼ 850 bases) to placental hCGα mRNA. RNA from butyrate-stimulated (5 mM, 24 h) ChaGo cells contained 7.7 times as much hCGα mRNA as RNA from control ChaGo cells. This increase appeared to be relatively selective since no difference in total polyA-containing mRNA levels was detected between butyrate-treated and control cells by [ 32P]oligo(dT) hybridization. In addition, no hCGβ mRNA was detected when Northern and dot blots were hybridized to an hCGβ cDNA probe. In a time course experiment, hCGα mRNA accumulation in butyrate-treated cells increased significantly by 8 h with a maximum increase of 6.1-fold at 24 h compared to control values. Major differences in immunoactive hCGα accumulation were not apparent, however, until after 24 h. These studies show that stimulation of ChaGo hCGα production by butyrate can be completely accounted for by pretranslational events and that failure to detect hCG or free hCGβ production by these cells is not due to poorly translatable RNA or post-translational protein degradation. Thus, exclusive ectopic production of only one of the hCG subunits is likely to be due to selective genomic expression.

428 THE ONTOOENY OF THE RESPONSE TO GROWTH HORMONE

We defined the ontogeny of five hepatic mRNA sequences which are responsive to growth hormone (GH) in juvenile (35 d) hypophysectomized rats ay in vitro translation of mRNA sequences and two-dimensional gel electrophoresis of the 35[S]-methionine labeled products. Each of the five had a well defined ontogeny which did not parallel the ontogeny of hepatic GH receptors or serum GH. Major periods for transition of this family of mRNA sequences were weaning and puberty. The pattern of ontogeny for two of the products was similar to that observed for GH responsive serum proteins. Induction of relative GH deficiency in rat pups by administration of propylthiouracil resulted in alterations in the mRNA sequences suggestive of GH deficiency; however, in twelve day old animals GH was much less effective in reversing these alterations than in older animals. Augmentation of normal pups with pituitary dependent hormones (thyroxine corticosterone and GH) resulted in changes consistent with an overall increase in maturation of the liver. This effect was also observed in non-hormonally responsive products, suggesting the change is not specific to the GH responsive mRNA family. From these observations we conclude that: 1). factors other than GH or GH receptor content are of importance in the expression of GH responsive products in the 12 day old animal; 2) the ontogeny of GH responsive serum proteins may reflect pretranslational events; 3) GH and the other pituitary dependent hormones modify the cadence of expression of hepatic gene products;4) and that the ability of the liver to respond to GH is an age dependent phenomenon.

Activation of a human T cell line: a two-stimulus requirement in the pretranslational events involved in the coordinate expression of interleukin 2 and gamma-interferon genes.

In the resting state, the T3-positive, human T cell line Jurkat does not synthesize detectable amounts of either interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Activation of Jurkat as measured by the secretion of substantial amounts of both lymphokines requires two distinct signals. One signal is produced by the phorbol ester, phorbol myristate acetate, and the other by either phytohemagglutinin or antibodies to T3. To elucidate the molecular events by which these activation signals lead to the synthesis of IL 2 and IFN-gamma activity we used cDNA probes to follow the appearance of IL 2 and IFN-gamma-specific transcripts after activation of Jurkat. These studies demonstrate that both signals are required for the appearance of IL 2 or IFN-gamma-specific transcripts and that the appearance of IL 2 and IFN-gamma RNA is coordinate with regard to a) the signals required for their production, b) the kinetics of their appearance, and c) the inhibition of their appearance by cyclosporin A. These studies suggest that distinct T cell-activation signals may operate through a common regulatory pathway involved in the expression of both IL 2 and IFN-gamma genes.