Presence Of Transcription Inhibitor Research Articles

Lost in Translation: Physiological Roles of Stored mRNAs in Seed Germination

Seeds characteristics such as germination ability, dormancy, and storability/longevity are important traits in agriculture, and various genes have been identified that are involved in its regulation at the transcriptional and post-transcriptional level. A particularity of mature dry seeds is a special mechanism that allows them to accumulate more than 10,000 mRNAs during seed maturation and use them as templates to synthesize proteins during germination. Some of these stored mRNAs are also referred to as long-lived mRNAs because they remain translatable even after seeds have been exposed to long-term stressful conditions. Mature seeds can germinate even in the presence of transcriptional inhibitors, and this ability is acquired in mid-seed development. The type of mRNA that accumulates in seeds is affected by the plant hormone abscisic acid and environmental factors, and most of them accumulate in seeds in the form of monosomes. Release of seed dormancy during after-ripening involves the selective oxidation of stored mRNAs and this prevents translation of proteins that function in the suppression of germination after imbibition. Non-selective oxidation and degradation of stored mRNAs occurs during long-term storage of seeds so that the quality of stored RNAs is linked to the degree of seed deterioration. After seed imbibition, a population of stored mRNAs are selectively loaded into polysomes and the mRNAs, involved in processes such as redox, glycolysis, and protein synthesis, are actively translated for germination.

Ribosomal protein pNO40 mediates nucleolar sequestration of SR family splicing factors and its overexpression impairs mRNA metabolism

The nucleolus acts as a key stress sensor and responds to changes in cellular growth rate and metabolic activity. In addition to its major role as the site of ribosome biogenesis, high-throughput proteomic analyses of purified nucleoli have highlighted the multi-functional nature of these organelles, and several SR family splicing factors, including SRSF1 and SRSF2, have been detected in human nucleolar proteome analysis. Here we provide evidence that pNO40, a 60s ribosomal protein associated with nucleoli, acts as a mediator for recruitment of SR family splicing factors into nucleoli. As a nucleolar protein, pNO40 was originally identified by yeast two-hybrid analysis as interacting with pnn, an SR-like protein involved in pre-mRNA splicing. To explore its functional interaction with pnn, we characterized the interplay between pNO40 and SR family proteins and demonstrated that pNO40 plays a role in recruiting SR splicing factors into the nucleoli. The targeting of pNO40 to the nucleoli is dependent on its extreme-carboxyl-terminus nuclear localization signals while the sequence at the amino-terminus of pNO40 enables its interaction with pnn. Nucleolar association of SR proteins results in defects in mRNA metabolism leading to global nuclear accumulation of poly(A)+ RNA and splicing defects. Animal studies confirmed aberrant mRNA splicing in transgenic muscles overexpressing pNO40 which displayed histological features of muscular dystrophy. Thus it appears that by pNO40 overexpression, we created mimics of nucleolar association of SR proteins occurring in the presence of transcription inhibitors which induce nucleolar segregation and redistribute SR proteins to the periphery of the nucleolar region. We therefore provide an extra-ribosomal function for pNO40 and, based on our data, it is conceivable that pNO40 may function as a general recruiter for nucleolar association of SR proteins and regulation of its expression may be crucial in cellular homeostasis.

Arginine methylation of hnRNPUL1 regulates interaction with NBS1 and recruitment to sites of DNA damage.

Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1RK. hnRNPUL1RK was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1RK had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1RK was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.

The Role of the 3′UTR Region in the Regulation of the ACVR1/Alk-2 Gene Expression

BackgroundThe ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3′UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression.ResultsThe ACVR1/Alk-2 3′UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3′UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3′UTR sequence thereby modulating its expression.ConclusionIn this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3′UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3′UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.

Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA.

The transcription factor TFIIA is encoded by two genes, TFIIAalphabeta and TFIIAgamma. In higher eukaryotes, the TFIIAalphabeta is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAbeta subunit starts at Asp 278. We found that a cleavage recognition site (CRS), a string of amino acids QVDG at positions -6 to -3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAbeta greatly affect degradation of the beta-subunit. We propose that cleavage and subsequent degradation fine-tune the amount of TFIIA in the cell and consequently the level of transcription.

Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.

Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell-expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription-PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)-regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type-specific genomic scale analyses of gene function.

Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution.

Cold-inducible RNA-binding protein (CIRP) was originally found in mammalian cells as a protein that is overexpressed upon a temperature downshift. Recently, we identified a Xenopus homolog of CIRP, termed xCIRP2, as a major cytoplasmic RNA-binding protein in oocytes. In this study we found by yeast two-hybrid screening that the Xenopus homolog of protein arginine N-methyltransferase 1 (xPRMT1) interacted with xCIRP2. We found that an arginine- and glycine-rich region of xCIRP2, termed the RG4 domain, was a target of xPRMT1 for methylation in vitro. xCIRP2 expressed in cultured cells accumulated in the nucleus as does mammalian CIRP. Interestingly, the RG4 domain was necessary for nuclear localization of xCIRP2. RG4-mediated nuclear accumulation of xCIRP2 was diminished in the presence of transcription inhibitors, suggesting that nuclear localization of xCIRP2 was dependent on ongoing transcription with RNA polymerase II. Analysis of interspecies heterokaryons revealed that xCIRP2 was capable of nucleocytoplasmic shuttling and the RG4 domain functioned as a nucleocytoplasmic shuttling signal. Methylation by overexpressed xPRMT1 caused cytoplasmic accumulation of xCIRP2. Possible implications of the relationship between regulation of intracellular localization and multiple functions of xCIRP2 will be discussed.

Cyclic AMP Prevents an Increase in GAP-43 but Promotes Neurite Growth in Cultured Adult Rat Dorsal Root Ganglion Neurons

High expression of the growth-associated protein GAP-43 in neurons is correlated with developmental and regenerative axon growth. It has been postulated that during development and after injury, GAP-43 expression is elevated due to the unavailability of a target-derived repressive signal, but that GAP-43 expression then declines upon target contact. Here we examine the cyclic AMP second messenger signaling pathway to determine if it might mediate retrograde transmission of a signal which represses GAP-43 expression and inhibits growth. Cultures of adult rat dorsal root ganglia were chronically exposed to membrane-permeable analogs of cyclic AMP and activators of adenyl cyclase. These treatments caused GAP-43 protein levels to decrease in a dose-dependent manner, although neuronal survival was not affected. GAP-43 mRNA was also decreases by cyclic AMP. GAP-43 protein levels were not repressed by neurotrophins, cytokines, or other agents. Surprisingly, cyclic AMP caused an increase in the rate of neurite outgrowth, even though the neurons were partially depleted of GAP-43. Growth stimulation was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern. These findings suggest that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP.

Homologous upregulation of gonadotropin-releasing hormone receptor mRNA occurs through transcriptional activation rather than modulation of mRNA stability.

In a previous study, we showed that even continuous application of gonadotropin-releasing hormone (GnRH) could increase the steady-state levels of GnRH receptor (GnRH-R) mRNA if treated for a relatively short period (6 h). Therefore, in the present study we examined whether GnRH-induced increment of GnRH-R mRNA is owing to stabilization of the preexisting GnRH-R mRNA or new synthesis of GnRH-R mRNA or both. Initially, to examine the effect on new RNA synthesis, the transcription inhibitor, actinomycin D (2 microM), was added to primary cultured rat anterior pituitary cells. In the presence of transcription inhibitor, GnRH-induced augmentation of GnRH-R mRNA levels was completely abolished. This result indicates that homologous upregulation of GnRH-R mRNA expression occurs at least through new RNA synthesis of GnRH-R gene. We further assessed the effects of GnRH on the turnover rate of GnRH-R mRNA using actinomycin D (2 microM). The basal half-life of GnRH-R mRNA was estimated to be approx 21 h. The application of GnRH tended to slightly suppress the basal turnover rate of GnRH; however, there was no statistically significant difference, compared with the group treated with actinomycin D alone. Collectively, our results suggest that the homologous upregulation of GnRH-R mRNA may occur through transcriptional activation of GnRH-R gene rather than enhancement of GnRH-R mRNA stability, although we did not examine the transcription rate of GnRH-R gene.

Pituitary adenylate cyclase activating polypeptide (PACAP) regulates expression of catecholamine biosynthetic enzyme genes in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) elevates levels of the mRNAs encoding the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in primary cultures of bovine adrenal chromaffin cells. PACAP potently (in nanomolar concentrations) increases the amount of mRNA for each of the three catecholamine biosynthetic enzymes. At 10 nM PACAP, TH and DBH mRNA levels increase approx 10-fold; 1 nM PACAP produces an approx 2.5-fold elevation of PNMT mRNA. In contrast to depolarizing or cholinergic stimuli, PACAP does not enhance expression of 5' upstream regions of the PNMT gene transiently transfected into chromaffin cells. Nor does PACAP stimulate the rate of PNMT gene transcription, thereby indicating that the effects of this neuropeptide do not involve enhanced transcription of this gene. However, after 16 h in the presence of transcriptional inhibitors, more PNMT mRNA is present in cultures treated with PACAP relative to control cultures, whereas amounts of TH and DBH mRNAs are not changed. PACAP likely elevates PNMT mRNA levels posttranscriptionally, possibly by stabilizing this message against degradation. Thus, although PACAP is an effective regulator for expression of all three catecholamine enzyme genes, its mechanism of action on PNMT mRNA appears to be distinctive from its effects on TH and DBH gene transcription.

Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage.

Global activation of the embryonic genome occurs at the 4- to 8-cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of transcripts for two paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20-24 h post-insemination in vitro and at intermediate stages up to the blastocyst stage. SRY transcripts were also detected at 2-cell to blastocyst stages. The expression of SRY and ZFY at these early stages and the faster cleavage rate of male embryos observed in many mammalian species focuses attention on the role of events in sex determination prior to gonad differentiation.

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin.

In primary cultures of rat pituitary cells, inhibin and follistatin reduce steady state levels of FSH beta mRNA to less than 10% of control within 4-6 h, while activin increases this mRNA 2- to 3-fold after 2-4 h of treatment. The effects of these three gonadal polypeptide hormones on the LH beta and common alpha-subunit mRNAs are more gradual and of lesser magnitude. The present study was designed to determine whether inhibin, activin, and/or follistatin act at the posttranscriptional level by altering the stability of the gonadotropin subunit mRNAs. To determine the decay rates of FSH beta, LH beta, and alpha-subunit mRNAs, primary pituitary cell cultures were treated for 1-24 h with either of two transcriptional inhibitors, actinomycin-D or 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB), in the presence or absence of recombinant human inhibin-A, recombinant human activin-A, or purified bovine follistatin. The decay of preexisting gonadotropin subunit mRNAs was followed by Northern blot analysis. Levels of LH beta and alpha-subunit mRNAs remained constant or increased during the 24-h exposure to transcriptional inhibitors; therefore, it was not possible to calculate their half-lives. The stability of these mRNAs was not altered by inhibin, activin, or follistatin. In contrast, FSH beta mRNA turned over rapidly: the estimated half-life was 2.6 +/- 0.19 h (mean +/- SEM of eight determinations) after actinomycin-D treatment and 1.9 +/- 0.14 h (mean +/- SEM of 12 determinations) after DRB treatment. When new RNA synthesis was blocked by either actinomycin-D or DRB, there were no significant effects of inhibin, activin, or follistatin on the stability of FSH beta mRNA (n = 2-4 for each hormone). The decay of FSH beta mRNA in the presence of inhibin or follistatin alone, however, was even more rapid than that determined after the administration of transcriptional inhibitors (P < 0.005). After an initial lag of 1-2 h, the half-life of FSH beta mRNA was 0.88 +/- 0.15 h (n = 4) or 0.62 +/- 0.11 h (n = 3), in the presence of inhibin or follistatin, respectively. The most likely interpretation of these results is that inhibin/follistatin reduces steady state levels of FSH beta mRNA by inducing a labile protein that accelerates the degradation of this mRNA species, and the synthesis of this protein is blocked by actinomycin-D or DRB treatment. It is not clear at present whether inhibin, follistatin, and activin have additional effects on transcription of the gonadotropin subunit genes.

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to greater than 95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptor-mediated pathway.

The effect of transcription inhibitors on early development of the avian embryo

The effect of transcription [actinomycin D, 5,6- dichloro-1-β- d - ribofuranosylbenzimidazole (DRB), α-amanitin] and translation inhibitors (cycloheximide, puromycin) on quail embryo development was investigated under in vitro conditions. The gastrulation process seemed to proceed normally in the presence of transcription inhibitors in the medium but the translation inhibitors stopped development and caused complete degeneration of the embryos.

Metabolic Control of Klebsiella pneumoniae mRNA Degradation by the Availability of Fixed Nitrogen

The chemical and functional stability of Klebsiella pneumoniae mRNAs was dependent on the nitrogen status of the organism. During exponential growth on N2 or NH+4 as N-source the half-lives of mRNAs were 10 and 4 min, respectively. When NH+4-grown cells were starved of NH+4 for 3 h, under which conditions the nitrogen fixation (nif) genes were derepressed, the half-life of mRNAs increased to 20 min. Addition of NH+4 to such N-starved suspensions rapidly destabilized mRNAs with a resultant half-life of 9 min. This destabilization occurred even in the presence of transcription inhibitors, which indicated metabolic control of mRNA degradation. Under most conditions studied the functional decay of nif-specific mRNAs paralleled the decay of bulk mRNAs.