Poly(A) Publications
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Poly(A) Publications
Luis Enrique Cabrera Quio, Alexander Schleiffer, Karl Mechtler, Andrea Pauli
Abstract
Post-transcriptional mechanisms are crucial for the regulation of gene expression. These mechanisms are particularly important during rapid developmental transitions such as the oocyte-to-embryo transition, which is characterized by dramatic changes to the developmental program in the absence of nuclear transcription. Under these conditions, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which cellular machineries contribute to remodeling the maternal transcriptome during the oocyte-to-embryo transition. Here, we focused on the auxiliary 3’-to-5’ degradation factor Ski7 in zebrafish as its mRNA peaks during this time frame. Homozygous ski7 mutant fish were viable and developed into morphologically normal adults, yet they had decreased fertility. Consistent with the idea that Ski7 participates in remodeling the transcriptome during the oocyte-to-embryo transition, transcriptome profiling identified stage-specific mRNA targets of Ski7. Genes upregulated in ski7 mutants were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. GO enrichment analyses of genes mis-regulated in ski7 mutants implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Overall, our results provide first insights into the physiological role of vertebrate Ski7 as an important post-transcriptional regulator during the oocyte-to-embryo transition.
Pavel Rossner, Jr., Kristyna Vrbova, Andrea Rossnerova, Tana Zavodna, Alena Milcova, Jiri Klema, Zbynek Vecera, Pavel Mikuska, Pavel Coufalik,Lukas Capka, Kamil Krumal, Bohumil Docekal, Vladimir Holan, Miroslav Machala and Jan Topinka
Abstract
We investigated the transcriptomic response and epigenetic changes in the lungs of mice exposed to inhalation of copper(II) oxide nanoparticles (CuO NPs) (8 × 105 NPs/m3) for periods of 3 days, 2 weeks, 6 weeks, and 3 months. A whole genome transcriptome and miRNA analysis was performed using next generation sequencing. Global DNA methylation was assessed by ELISA. The inhalation resulted in the deregulation of mRNA transcripts: we detected 170, 590, 534, and 1551 differentially expressed transcripts after 3 days, 2 weeks, 6 weeks, and 3 months of inhalation, respectively. Biological processes and pathways affected by inhalation, differed between 3 days exposure (collagen formation) and longer treatments (immune response). Periods of two weeks exposure further induced apoptotic processes, 6 weeks of inhalation affected the cell cycle, and 3 months of treatment impacted the processes related to cell adhesion. The expression of miRNA was not affected by 3 days of inhalation. Prolonged exposure periods modified miRNA levels, although the numbers were relatively low (17, 18, and 38 miRNAs, for periods of 2 weeks, 6 weeks, and 3 months, respectively). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis based on miRNA–mRNA interactions, revealed the deregulation of processes implicated in the immune response and carcinogenesis. Global DNA methylation was not significantly affected in any of the exposure periods. In summary, the inhalation of CuO NPs impacted on both mRNA and miRNA expression. A significant transcriptomic response was already observed after 3 days of exposure. The affected biological processes and pathways indicated the negative impacts on the immune system and potential role in carcinogenesis.
Features Poly(A) RNA Selection Kit and SENSE mRNA‐Seq Library Prep Kit for Ion Torrent
Jae Yoon Kim, Dae Yeon Kim, Youn-Jin Park, Myoung-Jun Jang
Abstract
Lentinula edodes is one of the most popular edible mushrooms worldwide and contains important medicinal components such as lentinan, ergosterol, and eritadenine. Mushroom metabolism is regulated by the mycelia and fruit body using light; however, in mushrooms, the underlying molecular mechanisms controlling this process as well as light-induced gene expression remain unclear. Therefore, in this study, we compared morphological changes and gene expression in the fruit bodies of L. edodes cultivated under blue light and continuous darkness. Our results showed that blue light primarily induced pileus growth (diameter and thickness) compared to dark cultivation. Alternatively, stipe length development was promoted by dark cultivation. We also performed RNAseq on L. edodes under the blue light/dark cultivation conditions. A total of 12,051 genes were used for aligning the Illumina raw reads and 762 genes that showed fold change cut-offs of >|2| and significance p-values of <0.05 were selected under blue light condition. Among the genes which showed two-fold changed genes, 221 were upregulated and 541 were downregulated. In order to identify blue light induced candidate genes, differentially expressed genes (DEGs) were selected according to 4-fold changes and validated by RT-PCR. We identified 8 upregulated genes under blue light condition, such as DDR48-heat shock protein, Fasciclin-domain-containing protein and carbohydrate esterase family 4 protein, FAD NAD-binding domain-containing protein that are involved in morphological development of primordium and embryonic muscle development, cell adhesion and affect the structure of cellulosic and non-cellulosic cell walls of fruit body development, and photoreceptor of blue light signaling for fruit body and pigment development, respectively. This study provides valuable insights into the molecular mechanisms underlying the role of blue light in mushroom growth and development and can thus contribute to breeding programs to improve mushroom cultivation.
Alessandro Scacchetti, Tamas Schauer, Alexander Reim, Zivkos Apostolou, Aline Campos Sparr, Silke Krause, Patrick Heun, Michael Wierer, Peter B. Becker
Abstract
Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene.
Features Poly(A) RNA Selection Kit
Soon Seok Kim, Jin Ah Lee, Min-Kyeong Yeo
Abstract
Hydra magnipapillata cells reduce the toxicity of silver nanomaterials to zebrafish (Danio rerio) embryos. In this study, we investigated whether Hydra protein (HP) and Hydra basal disc peptide (Hym176) materials reduce nano-Ag-polyvinylpyrrolidone (N-Ag-PVP) toxicity during embryogenesis of the nanosensitive organism zebrafish. Protein (HP) was extracted from Hydra, and peptide (Hym176) was extracted from the hydra basal disc, which is attractive to nanomaterials and related to the immune system. The experimental conditions were exposure to N-Ag-PVP, HP, N-Ag-PVP+HP, Hym176, or N-Ag-PVP+Hym176 during embryo development. N-Ag-PVP+HP group showed lower toxicity than N-Ag-PVP group. In addition, in the N-Ag-PVP+HP group formed aggregated nanomaterials (≥200 nm size) through electrostatic bonding. In the gene expression profile, HP group differed in gene expression profile compared the other experimental groups and it was no genetic toxicity. HP showed a tendency to reduce side effects and abnormal gene expression produced by N-Ag-PVP with no evidence of inherent toxicity. Considering the potential nanotoxicity effects of released nanomaterials on the ecosystem, the reduction of nanotoxicity observed with HP natural materials should be regarded with great interest in terms of the overall health of the ecosystem.
Features Poly(A) RNA Selection Kit and SENSE mRNA‐Seq Library Prep Kit for Ion Torrent
Joshua Young, Gina Zastrow-Hayes, Stéphane Deschamps, Sergei Svitashev, Mindaugas Zaremba, Ananta Acharya, Sushmitha Paulraj, Brooke Peterson-Burch, Chris Schwartz, Vesna Djukanovic, Brian Lenderts, Lanie Feigenbutz, Lijuan Wang, Clara Alarcon, Virginijus Siksnys, Gregory May, N. Doane Chilcoat & Sandeep Kumar
Abstract
CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.
Jacob Fleischmann, Miguel A. Rocha, Peter V Hausert
Abstract
While in eukaryotes three RNA polymerases are involved in ribosome production [1] under usual growth conditions, the 18S and 25S ribosomal RNA (rRNA) components are thought to be exclusively the products of transcription by RNA polymerase I (Pol I) followed by processing [2]. We have observed recently, in Candida albicans during nutritional depletion and with TOR inhibition, the appearance of 18S and 25S rRNA molecules, resisting digestion by a 5′-phosphate-dependent exonuclease, indicating that they were different from the usual processed rRNA transcripts [3]. Candida albicans, a eukaryotic yeast, is a major cause of invasive fungal disease especially in immune compromised patients [4]. Ribosomes of eukaryotic cells are assembled from four individual rRNAs and 79 proteins [5]. As in Saccharomyces cerevisiae, genes coding for rRNA (rDNA) in C. albicans are repeated multiple times in tandem [6], allowing for efficient transcription by Pol I. Like other eukaryotes, the current accepted mechanism of the production of the 18S and 25S components of the ribosome in this yeast, is transcription of a 35S copy of the rDNA, followed by post and co-transcriptional processing of the nascent RNA [7]. Typically, processed RNA molecules will have a single phosphate on their 5’-end making them vulnerable to processive 5′→3′ exonucleases (P53E) that digests only RNA that has a 5′-monophosphate end [8]. Therefore, after digestion by such an exonuclease, it was unexpected to find 18S and 25S rRNA molecules in total RNA isolated from C. albicans entering its stationary phase [3]. Similar molecules were appearing also in yeast, whose TOR was inhibited by rapamycin [3]. This background information is illustrated in Fig 1A. Pyrophosphatase digestion which separates linked phosphates, made these resistant 18S and 25S molecules vulnerable again to 5’-exonulease digestion [3]. This indicated that these molecules contained more than a single phosphate at their 5’-end. This in turn raised the possibility that they were newly transcribed rather than processed, as polymerases use triphosphate nucleotides when they initiate transcription. Another serial enzyme digestion included alkaline phosphatase (AP) followed by P53E digestion, in this case the rRNAs remained protected [3]. This could have been due to either more than one phosphate being digested by AP resulting in 5′-OH, or further modification in rRNA, such as a 5’-cap protecting against both AP and P53E, again preventing exonuclease digestion. Additionally, we have previously found that C. albicans grown overnight, polyadenylates some of its 18S [9] and 25S [10] rRNA molecules, a feature associated with Pol II transcription [11]. These features prompted us to see whether Pol II is involved with ribosomal rRNA transcription.
