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Argonaute proteins are key players in microRNA (miRNA) processing and function. Next to their role as RISC effector proteins mediating target silencing, they actively participate in miRNA biogenesis and increase miRNA abundance by a yet unidentified mechanism. It has been hypothesized that this increase in steady-state miRNA levels might be due to a stabilizing effect of Argonaute proteins, but this has not been analyzed so far due to a lack of test systems. Here, we provide two approaches to estimate miRNA stability and factors affecting it: in cells lacking Ago2, endogenous miRNA guide strand degradation and half-lives can be assessed using Actinomycin D or α-Amanitin. In turn, miRNA passenger strands can serve as a model in wildtype cells to ascertain the impact of miRNA stability factors. We provide evidence that Argonaute proteins stabilize mature miRNAs in a slicing-independent manner. Transcriptional inhibition reveals reduced half-lives of multiple endogenous miRNA guide strands in cells lacking Ago2. This effect is reversible upon the reconstitution of Argonaute expression. Correspondingly, overexpression of Argonaute proteins decelerates miRNA degradation and increases miRNA half-life. Taken together, this study employs two model systems to identify factors altering miRNA stability and provides evidence how Argonaute proteins post-transcriptionally elevate mature miRNA levels via increasing miRNA stability.

The ability of inhibitors of transcription and translation to prevent glucocorticoid-induced apoptosis has been interpreted to indicate that the cell death machinery requires de novo protein synthesis. The transcriptional inhibitors actinomycin D (Act D) and DRB as well as the translational inhibitors CHX and puromycin inhibited early loss of mitochondrial membrane integrity in a dose-dependent manner. This effect was not observed with the transcriptional inhibitor α-amanitin suggesting they may have additional effects. Their role in the glucocorticoid receptor (GR) intracellular trafficking was therefore investigated. Here, we show that Act D and CHX reduced glucocorticoid binding, GR turnover and impaired GR nuclear translocation. We performed the same experiments in different thymocyte subpopulations of Balb/c mice. At the highest dose tested, actinomycin D and cycloheximide abolished glucocorticoid-induced cell death of CD4+CD8+ and CD4+CD8-. In all subsets, Act D, DRB, as well as CHX and puromycin prevented receptor nuclear translocation, indicating a general alteration of GR trafficking. Overall, our data support a direct effect of macromolecular inhibitors on GR activation and trafficking. Finally, direct alterations of the functional properties of the glucocorticoid receptor might be responsible for cell death prevention by actinomycin D, DRB, cycloheximide and puromycin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

HOX proteins are transcription factors that play a major role in patterning the body axis of vertebrates from the gastrulation stage. While nothing has been reported so far about their roles at earlier stages, there is evidence that some HOX genes are expressed before gastrulation. The objective of this work was to study the pattern of expression of several HOX genes during oocyte maturation and early embryonic development up to the blastocyst stage. Using nested PCR, HOXD1, HOXA3, HOXD4, HOXB7, HOXB9, and HOXC9 transcripts were detected in bovine oocytes and early embryos at various frequencies depending on the stage of development. Quantitative PCR was performed on bovine oocytes and early embryos: relative expression of HOXD1, HOXA3, and HOXC9 decreased sharply after the 5-8 cell stage. HOXB9 relative expression increased between the oocyte and the morula stage. All transcripts seemed to be of maternal origin before the maternal to embryonic transition, as demonstrated by blocking transcription with α-amanitin. Reverse transcription was performed with either hexamers or oligo-dT, allowing for the determination that HOXC9 transcripts were slightly deadenylated during oocyte maturation; HOXD1, HOXA3, and HOXB9 transcripts were not, indicating that they could be translated. Hoxd1, Hoxa3, Hoxb9, and Hoxc9 expression was also detected in mouse oocytes and early embryos. A similar pattern of expression was found in the two species. In conclusion, mammalian HOX genes might be implicated in the control of oocyte maturation, the maternal-to-embryonic transition or the first steps of embryo differentiation., (Copyright © 2011 Wiley-Liss, Inc.)

RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis., Results: 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation., Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos ≥16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to ≥16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos ≥16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos ≥16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos ≥16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in ≥16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C(2) ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development.

Upon fertilization, the zygotic genome is activated. To ensure the transcription of specific genes and avoid promiscuous gene expression, a chromatin-mediated repressive state is established. To characterize potential heterochromatin factors present during the first cleavage, two putative transcriptional repressors, zinc finger protein (ZFP1) and ZFP54, belonging to the Kr&#x00FC;ppel-associated box (KRAB) zinc finger family, were isolated. ZFP1 and ZFP54 contain an N-terminally located KRAB repressor domain followed by 8 and 12 repeats of Kr&#x00FC;ppel zinc-finger motifs, respectively. Reverse transcription (RT) and quantitative (q) PCR show that maternally contributed Zfp1 and Zfp54 mRNA are detected throughout preimplantation development. &#x03B1;-Amanitin-treated zygotes revealed that maternal Zfp1 and Zfp54 are fully degraded at the two-cell stage. Microinjections of in vitro-transcribed mRNA encoding a gfp-fused reporter gene into zygotes demonstrated the intracellular distribution of ZFP1-green fluorescent protein (GFP) and ZFP54-GFP colocalized with a DNA marker in the two-cell embryo. The KRAB domain was essential to colocalize with DNA, and deletion of the KRAB domain in ZFP1-GFP and ZFP54-GFP localized in nucleoli and in a ubiquitously manner, respectively. Taken together, this suggests a role for ZFP1 and ZFP54 in transcriptional regulation in early development.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNA(Sec) however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNA(Sec) transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNA(Sec) is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNA(Sec) genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNA(Sec) outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNA(Sec), in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNA(Sec) gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatids.

Background: Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells., Methodology/principal Findings: To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After alpha-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense., Conclusions/significance: This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after alpha-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation.

Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using alpha-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression.

Objective: To investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus., Methods: A549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h., Results: The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly., Conclusion: DRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

Discrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase. The chromatin dynamics was not sensitive to transcription inhibition by alpha-amanitin but was significantly reduced by actinomycin D treatment. Since a majority of active genes replicate in early S-phase, our results suggest a correlation between levels of chromatin dynamics and chromatin poised for active transcription. Analysis of ChrD colocalization with transcription sites and cDNA with ChrD and transcription sites further supports this proposal.

MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

The aim was to test for evidence of transcriptional activity within the nuclei of the syncytiotrophoblast of the human placenta. The syncytiotrophoblast forms the epithelial covering of the villous tree, and is a multinucleated, terminally-differentiated syncytium generated through fusion of the underlying progenitor cytotrophoblast cells. Its nuclei are heterogeneous with respect to chromatin condensation, and previous functional studies of 3H-uridine uptake in vitro have indicated that they are transcriptionally inactive. This observation is surprising given the key roles this tissue plays in active transport, hormone synthesis and metabolic regulation, and has widespread implications for trophoblast physiology and pathophysiology. We used three different approaches to look for evidence of transcriptional activity. First, immunofluorescence staining was performed on paraffin-embedded early pregnancy and term placental villi, using an antibody directed specifically against the actively transcribing form of RNA polymerase II. Second, a nucleoside incorporation assay was applied to placental villi maintained in short-term culture, with and without the transcription blocker alpha-amanitin. Third, histone modifications associated with active chromatin were identified by immunohistochemistry and immunofluorescence. Each of these methods showed transcription to be occurring in a proportion of syncytiotrophoblast nuclei, with qualitative evidence for transcription being more abundant in the first trimester than at term. These findings correlated with electron microscopical observations of prominent nucleoli within the nuclei, particularly during early pregnancy, signifying transcription of ribosomal RNA. Contrary to previous findings, these results confirm that a proportion of syncytiotrophoblast nuclei actively produce mRNA transcripts.

MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3' untranslated regions of specific mRNAs and inhibit their translation. The correct regulation of mRNA expression by miRNAs is believed to be important for oocyte maturation, early development and implantation. We examined the expression of 25 mRNAs involved in the microRNA processing pathway in a nonhuman primate oocyte and embryo model. We observed that mRNAs related to miRNA splicing are downregulated during oocyte maturation while those related to miRNA processing are upregulated, indicating that there may exist a temporal difference in their activities related to transcriptional activity in germinal vesicle stage oocytes. We also observed that the vast majority of mRNAs examined were insensitive to alpha-amanitin at the 8-16 cell stage. The expression data did not reveal a major impact of embryo culture, and hormonal stimulation protocol affected only a small number of mRNAs, suggesting that the components of the pathway may be accumulated in the oocyte during oogenesis and resistant to exogenous insults. In comparison to published mouse array data, we observed species differences and similarities in the temporal expression patterns of some genes, suggesting that miRNA processing may be regulated differently. These data extend our understanding of the potential roles of miRNA during primate embryogenesis.

Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2-dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30-amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type-specific, these studies suggest that breast cancer cells contain a novel "death switch" that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis.

Purpose: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells., Materials and Methods: We introduced an alpha-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with alpha-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1., Results: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional., Conclusions: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.

The effect of pyriproxyfen, a juvenile hormone analog (JHA), on the pupation of S. litura was examined. A topical application of 100 mug JHA/larva on the newly ecdysed (0-day) sixth instar larvae resulted in more than 80% pupation, while most of the 1- or 2-day-old larvae similarly treated developed into supernumerary larvae. Glutathione S-transferse (GST) activity in the fat body of 0-day-old sixth instar larvae was significantly induced within 12 h of JHA (100 microg/larva) treatment. In contrast, no such induction was found when 1- and 2-day-old sixth instar larvae were similarly treated. This induction phenomenon was also observed when 0-day-old sixth instar larvae were treated with the natural JH III. The suppressive effects of alpha-amanitin and cycloheximide suggest that JHA induction of GST activity in these cutworm larvae presumably occurred at the gene transcription level., (Copyright 2008 Wiley-Liss, Inc.)

Background: Mutation of a tumor suppressor allele leaves the second as backup. Not necessarily so with p53. This homo-tetrameric transcription factor can become contaminated with mutant p53 through hetero-tetramerization. In addition, it can be out-competed by the binding to p53 DNA recognition motifs of transactivation-incompetent isoforms (DeltaN and DeltaTA-isoforms) of the p53/p63/p73 family of proteins. Countermeasures against such dominant-negative or dominant-inhibitory action might include the evolutionary gain of novel, transactivation-independent tumor suppressor functions by the wild-type monomer., Results: Here we have studied, mostly in human HCT116 colon adenocarcinoma cells with an intact p53 pathway, the effects of dominant-inhibitory p53 mutants and of Deltaex2/3p73, a tumor-associated DeltaTA-competitor of wild-type p53, on the nuclear transactivation-dependent and extra-nuclear transactivation-independent functions of wild-type p53. We report that mutant p53 and Deltaex2/3p73, expressed from a single gene copy per cell, interfere with the stress-induced expression of p53-responsive genes but leave the extra-nuclear apoptosis by mitochondrial p53 largely unaffected, although both wild-type and mutant p53 associate with the mitochondria. In accord with these observations, we present evidence that in contrast to nuclear p53 the vast majority of mitochondrial p53, be it wild-type or mutant, is consisting of monomeric protein., Conclusion: The extra-nuclear p53-dependent apoptosis may constitute a fail-safe mechanism against dominant inhibition.

Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.

As part of our systematic study of Korean toxic mushrooms, we have investigated the constituents of Amanita subjunquillea. The column chromatographic separation of the MeOH extract of A. subjunquillea led to the isolation of four ergosterols, two cerebrosides and four cyclopeptides. Their structures were determined by spectroscopic methods to be (22E,24R)-5alpha,8alpha-epidioxyergosta-6,9,22-triene-3beta-ol (1), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (2), (22E,24R)-5alpha,6alpha-epoxyergosta-8,22-diene-3beta,7beta-diol (3), (24S)-ergost-7-en-3beta-ol (4), 8,9-dihydrosoyacerebroside I (5), soyacerebroside I (6), beta-amanitin (7), phalloin (8), alpha-amanitin (9), and phalloidin (10). The compounds 1-6 and 8 were isolated for the first time from this mushroom. The isolated compounds were evaluated for the cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT15 cells. Compound 9 exhibited significant cytotoxic activity against A549, SK-OV-3, SK-MEL-2 and HCT15 with ED(50) values of 1.47, 0.26, 1.57 and 1.32 microM, respectively.

The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.

Centromeres are the fragments of DNA that are responsible for proper chromosome segregation. They consist of centromeric chromatin surrounded by blocks of pericentric heterochromatin, playing an important role in centromere function. In somatic cells, the pericentric domains have a specific pattern of epigenetic modifications of core histones and contain specific pericentric proteins. These features are probably more important for the centromere function than the sequence of the centromeric DNA itself. In somatic cells, the HP1alpha and HP1beta proteins are indispensable for constitutive heterochromatin formation and maintenance. We have analyzed the localization of these proteins in the primordial, growing, fully-grown, and maturing mouse oocytes. Additionally, we have analyzed post-translational modifications of histone H3, which can influence HP1alpha and HP1beta association with the heterochromatin. We showed that the regions of constitutive heterochromatin have a distinct pattern of histone H3 acetylation and di-, and trimethylation of its lysine 9. We demonstrated that HP1beta protein was present in pericentric chromatin domains in primordial oocytes, growing (transcriptionally active) oocytes, and in fully-grown oocytes, and was released to the cytoplasm after germinal vesicle breakdown. In contrast, the HP1alpha was never detected in primordial oocytes, was first detected in pericentric heterochromatin in growing oocytes, dissociated from pericentric heterochromatin in fully-grown oocytes, and it was never detected in maturing oocytes. The presence of HP1alpha and HP1beta proteins on the heterochromatin of transcriptionally active oocytes and their absence in transcriptionally silent oocytes suggest that they are necessary for the repression of RNA synthesis in heterochromatin domains of transcribing oocytes., (2007 Wiley-Liss, Inc)

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d.

Using two different mRNA synthesis inhibitors, we show that blockade of hippocampal gene expression during restricted posttraining or postretrieval time windows hinders retention of long-term spatial memory for the Morris water maze task, without affecting short-term memory, nonspatial learning, or the functionality of the hippocampus. Our results indicate that spatial memory consolidation induces the activation of the hippocampal transcriptional machinery and suggest the existence of a gene expression-dependent reconsolidation process that operates in the dorsal hippocampus at the moment of retrieval to stabilize the reactivated mnemonic trace., (2007 Wiley-Liss, Inc.)

Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

We have investigated the effect of alpha-amanitin treatment of mouse blastocysts on their further development. Twenty-four-hour treatments with alpha-amanitin at 1 microg/ml do not interfere with embryo survival or gross morphological development over the next few days although the rate of synthesis of polyA-containing RNA is dramatically reduced during exposure to the antimetabolite. Effects upon rates of protein synthesis are not observed initially, but incorporation of (35S)methionine into acid-insoluble material is ultimately inhibited by approximately 50% in all the treated samples. Developmental markers do not all respond in the same way to the alpha-amanitin treatments used. Whereas levels of beta-glucuronidase and plasminogen activator are virtually unaffected by the antimetabolite, alpha-amanitin treatment interferes with delta5,3beta-hydroxysteroid dehydrogenase activity in trophoblast cells and formation of a bilayered inner cell mass. The effects upon the latter markers differ quantitatively with the various exposure periods to alpha-amanitin. On the basis of these observations, we propose that mRNAs for at least some developmental markers in mouse blastocysts are synthesized long before they are translated.