Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis
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ABSTRACT:
ORGANISM(S): Karenia brevis
PROVIDER: GSE46175 | GEO | 2013/08/01
SECONDARY ACCESSION(S): PRJNA197497
REPOSITORIES: GEO
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Project description:Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into role of de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed RNA in Karenia brevis. These isolated fractions were then used for analysis of global de novo transcription by hybridization to a K. brevis microarray. As previous microarray studies indicated that transcripts for pentatricopeptide repeat (PPR) proteins rapidly increased in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of changes in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.M-bM-^@M-^C Cultures were acclimated to 10 M-BM-5M nitrate for a minimum of two months during which time cultures were transferred weekly in log phase. For addition experiments, nutrient replete and N-limited 1 L cultures were grown to stationary phase (Day 9). Using sodium nitrate, 155 M-BM-5M NO3 was added to stationary phase cultures. Cultures were exposed to 0.2 mM 4-thiouracil for 1 h to biosynthetically label newly transcribed RNA during the first hour post-N-addition (n=3) or during the fourth hour post-N-addition (n=3). N-limited cultures (n=3) were also exposed to 0.2 mM 4-thiouracil for 1 h to biosynthetically label newly transcribed RNA. Cultures were harvested after 1 h exposure to 4-thiouracil and total RNA extracted. Following extraction, RNA was biotinylated to allow for purification of the thiolated newly synthesized RNA from the total RNA pool using streptavidin coated magnetic beads. The newly transcribed RNA from each culture was independently labeled and hybridized to microarrys in a one color format. Based on the appearance of bioanlayzer profiles newly synthesized RNA was treated as mRNA in the labeling protocol.
2013-08-01 | E-GEOD-46175 | biostudies-arrayexpress
Project description:Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the role of differential mRNA stability in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed/pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 3 days, with a median of 33 hours. Thirteen percent of messages showed a half-life of 3 days, demonstrating their stability throughtout the course of the cell cycle and divison. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes.
2013-08-01 | GSE46174 | GEO
Project description:Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the role of differential mRNA stability in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed/pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 3 days, with a median of 33 hours. Thirteen percent of messages showed a half-life of 3 days, demonstrating their stability throughtout the course of the cell cycle and divison. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. Log phase cultures (n=6) were exposed to 0.2 mM 4-thiouracil for 2h to biosynthetically label newly transcribed RNA and total RNA was extracted. Following extraction, RNA was biotinylated to allow for purification of the thiolated newly synthesized RNA from the total RNA pool using streptavidin coated magnetic beads. From each replicate 3 pools of RNA, total RNA, pre-exisiting RNA, and newly synthesized RNA, were Cy3 labeled and hybridized to microarrys in a one color format. Based on the appearance of bioanlayzer profiles, total and pre-existing RNA were treated as total RNA, while newly synthesized RNA was treated as mRNA in the labeling protocol.
2013-08-01 | E-GEOD-46174 | biostudies-arrayexpress
Project description:The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is currently a contentious issue that warrants investigation into the nutrient physiology of this dinoflagellate species. The molecular mechanisms by which dinoflagellates respond to nutrient availability are essentially unexplored. The current study sought to determine if the transcriptome of K. brevis is responsive to nitrogen and informative of nutrient status. Stationary phase N-limited cultures did not exhibit up-regulation of transcripts considered to be hallmark of N depletion relative to nutrient-replete stationary phase cultures, despite rapid growth upon addition of the limiting nutrient. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. N-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p<10-4. By far, the earliest responding genes were dominated by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 4 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. Transcriptomic responses to the addition of N reflected primarily chloroplast functions. Even the earliest responding transcripts, those encoding PPR proteins, possess a 5’ trans-spliced leader sequence, suggesting that the observed rapid response of the transcriptome may be achieved, in part, through post-transcriptional mechanisms.
2011-08-09 | GSE28362 | GEO
Project description:The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is currently a contentious issue that warrants investigation into the nutrient physiology of this dinoflagellate species. The molecular mechanisms by which dinoflagellates respond to nutrient availability are essentially unexplored. The current study sought to determine if the transcriptome of K. brevis is responsive to phosphorus and informative of nutrient status. Stationary phase P-limited cultures did not exhibit up-regulation of transcripts considered to be hallmark of P depletion relative to nutrient-replete stationary phase cultures, despite rapid growth upon addition of the limiting nutrient. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p<10-4. By far, the earliest responding genes were dominated by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. Transcriptomic responses to the addition of P reflected primarily chloroplast functions. Even the earliest responding transcripts, those encoding PPR proteins, possess a 5’ trans-spliced leader sequence, suggesting that the observed rapid response of the transcriptome may be achieved, in part, through post-transcriptional mechanisms.
2011-08-09 | GSE28419 | GEO
Project description:Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis
| PRJNA197497 | ENA
Project description:Dinoflagellates have evolved a nuclear organization unlike that of any other eukaryotic group. Recent studies find a predominance of post-transcriptional control of dinoflagellate gene expression. This study investigated regulation of the environmental stress response in the red tide dinoflagellate, Karenia brevis using an Agilent custom oligonucleotide microarray. K. brevis cultures were exposed to 5°C or 10°C heat shock, or three different sources of oxidative stress: 60 µM H2O2, 10 mM NaNO2, or 12 µM PbCl2 over acute time courses. Ribosomal genes, genes involved in RNA processing, translation, and chaperones were among the classes of genes consistently downregulated across treatments, although within these functional classes the same genes did not always respond to different stressors. Genes involved in the photosystem and mitochondrial and chloroplast ATP generation dominated the down-regulated genes. Heat shock and oxidative stress response genes were not induced under any treatment, even under conditions that resulted in decreased viability. We subsequently identified the presence of a trans-spliced leader sequence on many stress response gene transcripts, which suggests that they may be transcribed constitutively and their expression regulated at the level of translation.
2010-12-01 | GSE13961 | GEO
Project description:Dinoflagellates have evolved a nuclear organization unlike that of any other eukaryotic group. Recent studies find a predominance of post-transcriptional control of dinoflagellate gene expression. This study investigated regulation of the environmental stress response in the red tide dinoflagellate, Karenia brevis using an Agilent custom oligonucleotide microarray. K. brevis cultures were exposed to 5°C or 10°C heat shock, or three different sources of oxidative stress: 60 µM H2O2, 10 mM NaNO2, or 12 µM PbCl2 over acute time courses. Ribosomal genes, genes involved in RNA processing, translation, and chaperones were among the classes of genes consistently downregulated across treatments, although within these functional classes the same genes did not always respond to different stressors. Genes involved in the photosystem and mitochondrial and chloroplast ATP generation dominated the down-regulated genes. Heat shock and oxidative stress response genes were not induced under any treatment, even under conditions that resulted in decreased viability. We subsequently identified the presence of a trans-spliced leader sequence on many stress response gene transcripts, which suggests that they may be transcribed constitutively and their expression regulated at the level of translation. Cultures of were grown to mid-log phase. For each treatment, five replicate untreated control cultures and five replicate treated cultures were harvested at several time points following treatment. The following time courses were used: 5°C heat shock - 5, 15, 30, 60, and 240 min; 10°C heat shock - 60 min; 60 µM H2O2 - 5, 15, 30, 60, and 240 min; 10 mM NaNO2 -1, 4, and 7 hours; 12 µM PbCl2 -1, 4, and 7 hours. For each treatment, RNA was pooled from the controls and treated cultures at each timepoint. Two color arrays were then run comparing each the transcriptome at timepoint with the pooled control for that treatment. A technical dye swap array was run at each timepoint.
2010-12-01 | E-GEOD-13961 | biostudies-arrayexpress
Project description:Karenia brevis Transcriptome or Gene expression
| PRJNA370691 | ENA
Project description:Transcriptional Responses of Karenia brevis to Acute Stresses
| PRJNA110289 | ENA