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Transcription and RNA decay determine steady-state RNA levels in cells available for translation and RNA-mediated regulatory functions. Both processes can be assessed by various techniques, for majority, based on RNA labelling or chromatin immunoprecipitation, but require a high level of expertise. Here, we describe a cost-effective, fast, and simple protocol that enables the profiling of nascent and mature RNA in the cytoplasm, nucleoplasm, and chromatin through subcellular fractionation. The workflow can include α-amanitin inhibition of RNA Polymerase II to assess nascent RNAs as a proxy of transcriptional activity, or it can be used without this treatment to investigate distribution of partially processed or mature transcripts across distinct subcellular compartments. It is applicable for studying any of RNA biotypes, including small and long noncoding RNAs, mRNAs, and their splice variants, on both transcript-specific and transcriptome-wide scales. Nascent or mature RNAs isolated from each fraction can be further analyzed by any technique of choice (northern blot, reverse transcription, RNA sequencing)., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA polymerase II drives mRNA gene expression, yet our understanding of Pol II degradation is limited. Using auxin-inducible degron, we degraded Pol II's RPB1 subunit, resulting in global repression. Surprisingly, certain genes exhibited increased RNA levels post-degradation. These genes are associated with GPCR ligand binding and are characterized by being less paused and comprising polycomb-bound short genes. RPB1 degradation globally increased KDM6B binding, which was insufficient to explain specific gene activation. In contrast, RPB2 degradation repressed nearly all genes, accompanied by decreased H3K9me3 and SUV39H1 occupancy. We observed a specific increase in serine 2 phosphorylated Pol II and RNA stability for RPB1 degradation-upregulated genes. Additionally, α-amanitin or UV treatment resulted in RPB1 degradation and global gene repression, unveiling subsets of upregulated genes. Our findings highlight the activated transcription elongation and increased RNA stability of signaling genes as potential mechanisms for mammalian cells to counter RPB1 degradation during stress., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

α-Amanitin, the primary lethal toxin of Amanita, specifically targets the liver, causing oxidative stress, hepatocyte apoptosis, and irreversible liver damage. As little as 0.1 mg/kg of α-amanitin can be lethal for humans, and there is currently no effective antidote for α-amanitin poisoning. Cannabidiol is a non-psychoactive natural compound derived from Cannabis sativa that exhibits a wide range of anti-inflammatory, antioxidant, and anti-apoptotic effects. It may play a protective role in preventing liver damage induced by α-amanitin. To investigate the potential protective effects of cannabidiol on α-amanitin-induced hepatocyte apoptosis and oxidative stress, we established α-amanitin exposure models using C57BL/6J mice and L-02 cells in vitro. Our results showed that α-amanitin exposure led to oxidative stress, apoptosis, and DNA damage in both mouse hepatocytes and L-02 cells, resulting in the death of mice. We also found that cannabidiol upregulated the level of Nrf2 and antioxidant enzymes, alleviating apoptosis, and oxidative stress in mouse hepatocytes and L-02 cells and increasing the survival rate of mice. Our findings suggest that cannabidiol has hepatoprotective effects through the regulation of Nrf2 and antioxidant enzymes and may be a potential therapeutic drug for Amanita poisoning., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita . Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 μg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.

Data normalization is critical to the process of estimating RNA degradation by analyzing RNA levels when transcription is blocked. Here, we present a protocol for measuring mRNA degradation rates, optimized for mouse embryonic stem cells, using α-amanitin inhibitor. We describe steps for a time course α-amanitin treatment, RNA-seq, and alignment; we then detail procedures for analyzing data and sequence enrichment. Our method relies on large-scale normalization of stable transcripts in genomic RNA-seq measurements, providing reliable readouts. For complete details on the use and execution of this protocol, please refer to Viegas et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The "death cap", Amanita phalloides, is the world's most poisonous mushroom, responsible for 90% of mushroom-related fatalities. The most fatal component of the death cap is α-amanitin. Despite its lethal effect, the exact mechanisms of how α-amanitin poisons humans remain unclear, leading to no specific antidote available for treatment. Here we show that STT3B is required for α-amanitin toxicity and its inhibitor, indocyanine green (ICG), can be used as a specific antidote. By combining a genome-wide CRISPR screen with an in silico drug screening and in vivo functional validation, we discover that N-glycan biosynthesis pathway and its key component, STT3B, play a crucial role in α-amanitin toxicity and that ICG is a STT3B inhibitor. Furthermore, we demonstrate that ICG is effective in blocking the toxic effect of α-amanitin in cells, liver organoids, and male mice, resulting in an overall increase in animal survival. Together, by combining a genome-wide CRISPR screen for α-amanitin toxicity with an in silico drug screen and functional validation in vivo, our study highlights ICG as a STT3B inhibitor against the mushroom toxin., (© 2023. The Author(s).)

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.

Key Message: The moss Pseudocrossidium replicatum is a desiccation-tolerant species that uses an inducible system to withstand severe abiotic stress in both protonemal and gametophore tissues. Desiccation tolerance (DT) is the ability of cells to recover from an air-dried state. Here, the moss Pseudocrossidium replicatum was identified as a fully desiccation-tolerant (FDT) species. Its gametophores rapidly lost more than 90% of their water content when exposed to a low-humidity atmosphere [23% relative humidity (RH)], but abscisic acid (ABA) pretreatment diminished the final water loss after equilibrium was reached. P. replicatum gametophores maintained good maximum photosystem II (PSII) efficiency (Fv/Fm) for up to two hours during slow dehydration; however, ABA pretreatment induced a faster decrease in the Fv/Fm. ABA also induced a faster recovery of the Fv/Fm after rehydration. Protein synthesis inhibitor treatment before dehydration hampered the recovery of the Fv/Fm when the gametophores were rehydrated after desiccation, suggesting the presence of an inducible protective mechanism that is activated in response to abiotic stress. This observation was also supported by accumulation of soluble sugars in gametophores exposed to ABA or NaCl. Exogenous ABA treatment delayed the germination of P. replicatum spores and induced morphological changes in protonemal cells that resembled brachycytes. Transcriptome analyses revealed the presence of an inducible molecular mechanism in P. replicatum protonemata that was activated in response to dehydration. This study is the first RNA-Seq study of the protonemal tissues of an FDT moss. Our results suggest that P. replicatum is an FDT moss equipped with an inducible molecular response that prepares this species for severe abiotic stress and that ABA plays an important role in this response., (© 2021. The Author(s).)

Alpha-amanitin, one of the amatoxins in egg amanita, has a cyclic peptide structure, and was reported as having antiviral activity against several viruses. We investigated whether α-amanitin has antiviral activity against feline immunodeficiency virus (FIV). FL-4 cells persistently infected with FIV Petaluma were cultured with α-amanitin. Reverse transcriptase (RT) activity in the supernatant of FL-4 cells was significantly inhibited by α-amanitin. In addition, the production of FIV core protein in FL-4 cells was inhibited by α-amanitin when analyzed by western blotting. Furthermore, α-amanitin inhibited the transcription of FIV in real-time RT-PCR. These data suggested that α-amanitin showed anti-FIV activity by inhibiting the RNA transcription level.

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Despite the great success of small molecule inhibitors in the treatment of patients with BRAF V600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAF V600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAF V600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAF V600E inhibitors.

Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression., Abbreviations: ANOVA: analysis of the variance; DMEM: Dulbecco's modified Eagle's medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor.

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors., Competing Interests: Conflict-of-interest statement: The authors declare no competing financial interests.

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2 S,3 R,4 R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target., (© 2018 Liu et al.)

H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To study the relationship between chromatin condensation, gene transcription and developmental competence during oocyte maturation and to explore the mechanisms by which meiotic arrest maintenance (MAM) and sexual maturity improve oocyte competence, we examined effects of MAM with roscovitine or db-cAMP on chromatin condensation, gene transcription and developmental potential of NSN or SN oocytes from prepubertal or adult mice. MAM with roscovitine improved the developmental competence and global gene transcription of prepubertal NSN (prep-NSN) and adult-SN oocytes while having no effect on those of prep-SN oocytes. MAM with db-cAMP facilitated neither development nor transcription in any type of oocytes. MAM with either roscovitine or db-cAMP promoted chromatin condensation of prep-NSN oocytes. MAM with roscovitine promoted gene transcription and chromatin condensation simultaneously through inhibiting cyclin-dependent kinase (CDK) 5 and 2, respectively. The results suggested that MAM with roscovitine improved oocyte competence by promoting gene transcription via inhibiting CDK5. Oocyte cytoplasmic maturation is correlated with gene transcription but not with chromatin condensation. The difference in developmental competence between prepubertal NSN and SN oocytes and between prepubertal and adult SN oocytes was because while the former had not, the latter had completed or acquired the ability for transcription of important genes.

Reactivated memories can be modified during reconsolidation, making this process a potential therapeutic target for posttraumatic stress disorder (PTSD), a mental illness characterized by the recurring avoidance of situations that evoke trauma-related fears. However, avoidance memory reconsolidation depends on a set of still loosely defined boundary conditions, limiting the translational value of basic research. In particular, the involvement of the hippocampus in fear-motivated avoidance memory reconsolidation remains controversial. Combining behavioral and electrophysiological analyses in male Wistar rats, we found that previous learning of relevant nonaversive information is essential to elicit the participation of the hippocampus in avoidance memory reconsolidation, which is associated with an increase in theta- and gamma-oscillation power and cross-frequency coupling in dorsal CA1 during reactivation of the avoidance response. Our results indicate that the hippocampus is involved in memory reconsolidation only when reactivation results in contradictory representations regarding the consequences of avoidance and suggest that robust nesting of hippocampal theta-gamma rhythms at the time of retrieval is a specific reconsolidation marker. SIGNIFICANCE STATEMENT Posttraumatic stress disorder (PTSD) is characterized by maladaptive avoidance responses to stimuli or behaviors that represent or bear resemblance to some aspect of a traumatic experience. Disruption of reconsolidation, the process by which reactivated memories become susceptible to modifications, is a promising approach for treating PTSD patients. However, much of what is known about fear-motivated avoidance memory reconsolidation derives from studies based on fear conditioning instead of avoidance-learning paradigms. Using a step-down inhibitory avoidance task in rats, we found that the hippocampus is involved in memory reconsolidation only when the animals acquired the avoidance response in an environment that they had previously learned as safe and showed that increased theta- and gamma-oscillation coupling during reactivation is an electrophysiological signature of this process., (Copyright © 2017 the authors 0270-6474/17/379675-11$15.00/0.)

Early embryos of the clitellate annelid Tubifex (oligochaete) undergo a series of unequal spiral cell divisions before the descendants of the D quadrant micromeres (cells 2d and 4d) divide bilaterally. Here, we show that inhibition of zygotic transcription by microinjection of α-amanitin (transcription inhibitor) exclusively converts unequal cleavage in cell 2d 11 (granddaughter of 2d) into equal cleavage while other unequal cleavages and ensuing bilateral cleavages in cells 4d and 2d 111 (great-granddaughter of 2d) all proceed in a normal fashion in the presence of this inhibitor. These results differ significantly from those reported for embryos of another clitellate annelid Helobdella (leech), in which inhibition of transcription converts bilateral (symmetric) cleavages in cells DNOPQ"' and DM" (equivalent to 2d 111 and 4d) into unequal (asymmetric) cleavages while having no apparent effect on unequal cleavage in DNOPQ" (equivalent to 2d 11 ). These differences imply distinct mechanisms for the control of the unequal-to-bilateral transition in the two clitellate annelids.

We investigated the mechanisms of mushroom toxin resistance in the Drosophila Genetic Reference Panel (DGRP) fly lines, using genome-wide association studies (GWAS). While Drosophila melanogaster avoids mushrooms in nature, some lines are surprisingly resistant to α-amanitin-a toxin found solely in mushrooms. This resistance may represent a pre-adaptation, which might enable this species to invade the mushroom niche in the future. Although our previous microarray study had strongly suggested that pesticide-metabolizing detoxification genes confer α-amanitin resistance in a Taiwanese D. melanogaster line Ama-KTT, none of the traditional detoxification genes were among the top candidate genes resulting from the GWAS in the current study. Instead, we identified Megalin, Tequila, and widerborst as candidate genes underlying the α-amanitin resistance phenotype in the North American DGRP lines, all three of which are connected to the Target of Rapamycin (TOR) pathway. Both widerborst and Tequila are upstream regulators of TOR, and TOR is a key regulator of autophagy and Megalin-mediated endocytosis. We suggest that endocytosis and autophagy of α-amanitin, followed by lysosomal degradation of the toxin, is one of the mechanisms that confer α-amanitin resistance in the DGRP lines.

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.

Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.

Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.

Transcription termination for genes encoding polyadenylated mRNAs requires a functional poly(A) signal (PAS) in the nascent pre-mRNA. Often called PAS-dependent termination, or PADT, it is widely assumed that the PAS requirement reflects an obligatory poly(A) site cleavage requirement for termination. Cleavage is thought to provide entry for a 5'-to-3' exonuclease that targets RNA polymerase II via the nascent transcript-i.e., the torpedo model. To assess the role of cleavage in PADT, we developed a PADT assay using HeLa nuclear extract. Here we examine the basal mechanism of PADT and show that cleavage at the poly(A) site is not required for PADT. Isolated elongation complexes undergo termination in a PAS-dependent manner when incubated in buffer, in the absence of extract, nucleotides, or cleavage at the poly(A) site. Thus, PADT-proficient complexes undergo a conformational change that triggers termination. PADT is inhibited by α-amanitin, which presumably blocks the required conformational change., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Object recognition memories (ORM) can incorporate new information upon reactivation. This update initially involves destabilization of the original memory, which is followed by restabilization of the upgraded engram through a reconsolidation process that requires gene expression and protein synthesis in the hippocampus. We found that when given in dorsal CA1 either immediately after training or 15 min before ORM reactivation in the presence of a novel object, the dopamine D1/D5 receptor antagonist SCH23390 did not affect ORM consolidation, expression or retention but impeded the amnesia caused by the post-retrieval administration of the mRNA synthesis inhibitor α-amanitin or the protein synthesis blocker anisomycin. This anti-amnesic effect was not observed when SCH23390 was given immediately after training and again 15 min before memory reactivation. Our results demonstrate that hippocampal D1/D5 receptors are not needed for formation, retrieval or post-retrieval restabilization of the ORM trace but are essential for its destabilization when reactivation occurs together with the incorporation of new information into the original memory. Importantly, they also suggest that reenactment of the animal's post-learning neurochemical milieu at the moment of memory reactivation can be a boundary condition for reconsolidation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of ∼ 6,000 intronic lncRNAs and ∼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent., (© 2014 by the Society for the Study of Reproduction, Inc.)

Background: We previously demonstrated embryotrophic actions of maternal (oocyte-derived) follistatin during bovine early embryogenesis. Classical actions of follistatin are attributed to inhibition of activity of growth factors including activins and bone morphogenetic proteins (BMP). However, temporal changes in BMP mRNA in early bovine embryos and the effects of exogenous BMP on embryo developmental progression are not understood. The objectives of present studies were to characterize mRNA abundance for select BMP, BMP receptors and BMP receptor associated SMADs during bovine oocyte maturation and early embryogenesis and determine effects of addition of exogenous BMP protein on early development., Methods: Relative abundance of mRNA for BMP2, BMP3, BMP7, BMP10, SMAD1, SMAD5, ALK3, ALK6, ALK2, BMPR2, ACVR2A and ACVR2B was determined by RT-qPCR analysis of germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes and in vitro produced embryos collected at pronuclear, 2-cell (C), 4C, 8C, 16C, morula and blastocyst stages. Effects of addition of recombinant human BMP2 (0, 1, 10 and 100 ng/ml) during initial 72 h of embryo culture on early cleavage (within 30 h post insemination), total cleavage, development to 8C-16C and blastocyst stages and blastocyst mRNA abundance for markers of inner cell mass (NANOG) and trophectoderm (CDX2) were also determined., Results: Abundance of mRNA for BMP2, BMP10, SMAD1, SMAD5, ALK3, ALK2, BMPR2 and ACVR2B was elevated in MII oocytes and/or pronuclear stage embryos (relative to GV) and remained elevated through the 8C -16C stages, whereas BMP3, BMP7 and ALK2 mRNAs were transiently elevated. Culture of embryos to the 8C stage in the presence of α-amanitin resulted in increased abundance for all of above transcripts examined relative to untreated 8C embryos. Effects of addition of exogenous BMP2 on early cleavage rates and rates of development to 8C-16C and blastocyst stages were not observed, but BMP2 treatment increased blastocyst mRNA for CDX2 and NANOG., Conclusions: Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis.

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mechanisms of induction and retrieval of latent (hidden) memory for conditioned food aversion were investigated in snails. After initial training (single combination of a food stimulus with electric shock), aversive reactions to presentation of the conditioned food stimulus were not revealed. Repeated presentation of the stimuli in 12 days after the first combination was followed by the appearance of aversive food reactions that persisted for at least 14 days. Injections of inhibitors of protein (cycloheximide) or RNA (α-amanitin) synthesis immediately after the first or second combined presentation of the stimuli disturbed skill performance. We hypothesized that single combination of food and reinforcing stimuli led to translation- and transcription-dependent induction of latent conditioned food aversion memory. Transformation of this memory into an active state after repeated presentation of the stimulus combination also depends on the synthesis of new proteins and RNA.

We have previously shown the differential expression of versican in the mouse uterus under ovarian hormone influence. We also demonstrated there is not a direct correlation between mRNA levels and protein expression, suggesting posttranscriptional events, such as alteration in mRNA stability. This posttranscriptional effect may result in the elongation and stabilization of transcripts poly(A) tail. Thus, the aim of this study was to analyze whether estradiol (E2) regulates versican mRNA stability and expression in a dose-related and time-dependent manner. For this purpose female mice were ovariectomized and treated with a single injection of 0.1 or 10 μg E2. To block transcription a group of females received a single injection of alpha-amanitin before hormone administration. Uterine tissues were collected 30 min, 1, 3, 6, 12 and 24 h after treatments and processed for quantitative real time PCR (qPCR), RACE-PAT Assay and immunohistochemistry. qPCR showed that versican mRNA levels are higher than control from 3 to 24 h after E2 administration, whereas after transcription inhibition versican mRNA unexpectedly increases within 3 h, which can be explained when transcriptional blockers alter the degradation rate of the transcript, resulting in the superinduction of this mRNA. Accordingly, analysis of versican transcript poly(A) tail evidenced a longer product 3 h after treatment, but not after 12 h. Versican immunoreaction becomes conspicuous in the superficial stroma only 3 h after E2 injection, whereas the whole stroma is immunoreactive from 6 h onward. These results demonstrate that E2 modulates versican at the transcriptional and posttranscriptional levels in a time-dependent manner.

Toxins could be effective anticancer drugs, if their selective delivery into cancer cells could be achieved. We have shown that the energy of membrane-associated folding of water-soluble membrane peptides of the pHLIP (pH low insertion peptide) family could be used to move cell-impermeable cargo across the lipid bilayer into the cytoplasm of cancer cells. Here we present the results of a study of pHLIP-mediated cellular delivery of a polar cell-impermeable toxin, α-amanitin, an inhibitor of RNA polymerase II. We show that pHLIP can deliver α-amanitin into cells in a pH-dependent fashion and induce cell death within 48 h. Translocation capability could be tuned by conjugating amanitin to the C-terminus of pHLIP via linkers of different hydrophobicities that could be cleaved in the cytoplasm. pHLIP-SPDP-amanitin, which exhibits 4-5 times higher antiproliferative ability at pH 6 than at pH 7.4, was selected as the best construct. The major mechanism of amanitin delivery is direct translocation (flip) across a membrane by pHLIP and cleavage of the S-S bond in the cytoplasm. The antiproliferative effect was monitored on four different human cancer cell lines. pHLIP-mediated cytoplasmic delivery of amanitin could create great opportunities to use the toxin as a potent pH-selective anticancer agent, which predominantly targets highly proliferative cancer cells at low extracellular pH values.

Old and young donors cells show different ability to homologous recombination (shown on the first stage--the chromosome transference) in vitro, that we suppose could be the reasons of the genome instability in aging. Homologous recombination, induced by X-radiation, is limited in cells taken from donors older than 70 years. Alpha-amanitin, the RNA-polymerize II repressor, in toxic doze, could induce the chromosome transference in the cells from all studied groups: from old and young donors and donors with repair process defect (with BRCA 1, 2 mutations). Summarized effect of X-radiation and alpha-amanitin does not increase the induction of the chromosome transference.

In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases Pol IV and Pol V and the putative RNA-dependent RNA polymerase RDR2. Here we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to α-amanitin, and differ in their ability to displace the nontemplate DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is nonfunctional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5'-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.

In animals, the maternal-to-embryonic transition (MET) occurs in the first days of early development and involves the degradation of maternal transcripts that have been stored during oogenesis. Moreover, precise and specific control mechanisms govern the adequate synchronization of the MET events to promote the activation of the embryonic genome. These mechanisms are not well understood, but it is believed that microRNAs (miRNAs) could be one of the mechanisms involved. After a microarray screening study, we analysed the expression of specific miRNA during oocyte maturation and early embryo development until preimplantation stages. Two differentially expressed candidates were selected for further analysis. Mature and precursor forms of miR-21 and miR-130a were quantified by qRT-PCR in pools of 20 oocytes at GV (germinal vesicle), GV breakdown and metaphase II stages as well as in pools of embryos at the 2-cell, 4-cell, 8-cell and blastocyst stages. The results showed a linear increase during the 1-8 cell stage for the mature forms of miR-130a and miR-21 (P < 0.05 and P < 0.003, respectively) and for the precursor form of miR-130a (P < 0.002). To see if this increase was due to minor transcriptional activity, 2-cell embryos were exposed to α-amanitin for 30-34 h. Results showed a significant decrease in miR-21, pre-miR-21, miR-130a and SRFS3 in α-amanitin-treated embryos (P < 0.05). Considering the potential regulatory role of these miRNA, the bovine genome was screened to identify putative targets with a 3'UTR exact seed match. This study suggests that miRNAs could be important players in the MET, as expression profiles suggest a potential regulation role during early development steps.

Memory is destabilized during retrieval-induced reconsolidation and can therefore be disrupted or modified. In the present study, we examined the role of translation and transcription processes in long-term food aversion memory reconsolidation in the snail Helix lucorum. The administration of the protein synthesis inhibitor anisomycin followed by a reminding procedure (presentation of the conditioned stimulus) led to the development of amnesia that persist for 2 weeks or longer. Administration of the mRNA synthesis inhibitors actinomycin D, α-amanitin, or DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidasole) followed by a reminding procedure did not affect memory retrieval. Our present findings indicate that proteins synthesized from preexisting mRNA that is transcribed during learning and stored in a silent state may be involved in the mechanisms of conditioned food aversion reconsolidation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus implicated in lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma, and NK/T cell lymphoma. MicroRNAs (miRNAs) are 19-25 nucleotide long single-stranded RNAs involved in post-transcriptional gene regulation. miRNAs are mainly transcribed by RNA polymerase II (pol II) to have stem-loop structures and subsequently processed by Drosha and Dicer. EBV miRNAs are expressed in B cells, nasopharyngeal carcinoma cells, and gastric carcinoma cells infected with EBV. EBV miRNAs can be divided into two groups: BHRF1 miRNAs and BART miRNAs. In this study, we investigated the biogenesis of EBV miRNAs. Treatment of the SNU-719 EBV-positive gastric cancer cell line with α-amanitin at a concentration that selectively inhibits RNA polymerase II activity decreased the expression levels of BART miRNAs. The expression levels of BART miRNAs were also reduced by RNA interference targeting Drosha and Dicer. Two of each C/EBPβ and c-Myc binding sites are located upstream of the proposed initiation sites for primary BART miRNA transcripts. Knockdown of C/EBPβ but not c-Myc using siRNAs reduced BART miRNA expression by 25-55% compared with the control. These results suggest that BART miRNAs are transcribed by pol II and undergo a similar biogenesis process with cellular miRNAs.

Embryonic genome activation (EGA) is the first major step towards successful initiation of preimplantation development, which culminates in the formation of implantation-competent embryos. EGA occurs at species-specific embryonic cell stages. In the present work, EGA was identified for buffalo embryos by studying the development rate of embryos in normal as well as imposed transcription block conditions, analyzing bromo-uridine triphosphate (BrUTP) incorporation rates as evidence of de novo transcription initiation, and studying the expression status of eukaryotic translation initiation factor 1A (eIF1A), U2 auxiliary splicing factor (U2AF), and polyadenylate polymerase (PAP) genes at different embryonic cell stages. Under normal, in vitro fertilization and culture conditions, about 26% and 17% of oocytes could reach morula and blastocyst stages, respectively, but no embryos could progress beyond 8-cell stages in presence of α-amanitin. Culturing embryos in the presence of BrUTP revealed a marked increase in its incorporation between 4- and 8-cell stages. All genes studied displayed an abrupt increase in expression between 4- and 8-cell stages; PAP expression was upregulated earlier from 2- to 4-cell stages. About 65% of PAP transcripts from the 4-cell stage and more than 70% of eIF1A, U2AF, and PAP transcripts at 8-cell stage embryos were found to be synthesized de novo. Together, these data suggest that a minor EGA in buffalo embryos happens from 2- to 4-cell stages, while the major EGA takes place from 4- to 8-cell stage transition., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects., Methods: Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided., Results: In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 μg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 μg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama., Conclusion: This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to α-amanitin. Mutations of interest are introduced into an α-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with α-amanitin. Here, we show that cells that are enabled to grow in α-amanitin by expression of an α-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an α-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in α-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of α-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in α-amanitin, presumably due to the α-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the α-amanitin-resistant Rpb1-containing polymerase.

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

During the final stage of oocyte growth, the morphology of the oocyte nucleoli changes into a compact structure. The objective of this study was to determine the involvement of the proteasome, which is a large protein complex responsible for degrading intracellular proteins, in the nucleolar compaction. The mean nucleolar diameter of growing porcine oocytes (about 100 µm in diameter) was larger than that of fully grown (120 µm) oocytes (15.5 ± 0.3 vs. 13.2 ± 0.1 µm, P<0.05). When fully grown oocytes were treated with proteasome inhibitors, MG132 (10 and 20 µM) and lactacystin (100 and 200 µM), the nucleolar diameter significantly increased from 12.9 µm to 14.9-16.1 µm. In contrast, transcription inhibitors, actinomycin D (0.8-8 µM) and α-amanitin (10-100 µM) reduced the nucleolar diameter of growing oocytes to 9.4-12.4 µm. MG132 partially prevented this reduction in nucleolar diameter. These results suggest that the proteasome regulates the nucleolar size in porcine oocytes perhaps through the degradation of nucleolar proteins.

Many mycophagous species of Drosophila can tolerate the mushroom poison α-amanitin in wild mushrooms and in artificial diet. We conducted feeding assays with sixteen Drosophila species and α-amanitin in artificial diet to better determine the phylogenetic distribution of this tolerance. For eight tolerant and one related susceptible species, we sequenced the gene encoding the large subunit of RNA Polymerase II, which is the target site of α-amanitin. We found no differences in the gene that could account for differences in susceptibility to the toxin. We also conducted feeding assays in which α-amanitin was combined with chemical inhibitors of cytochrome P450s or glutathione S-transferases (GSTs) in artificial diet to determine if either of these enzyme families is involved in tolerance to α-amanitin. We found that an inhibitor of GSTs did not reduce tolerance to α-amanitin, but that an inhibitor of cytochrome P450s reduced tolerance in several species. It is possible that the same cytochrome P450 activity that produces tolerance of α-amanitin might produce tolerance of other mushroom toxins as well. If so, a general detoxification mechanism based on cytochrome P450s might answer the question of how tolerance to α-amanitin arose in mycophagous Drosophila when this toxin is found in relatively few mushrooms.