Unknown,Transcriptomics,Genomics,Proteomics

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Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis

ABSTRACT: 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.

ORGANISM(S): Karenia brevis

SUBMITTER: Frances Van Dolah

PROVIDER: E-GEOD-46175 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 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.

Project description:During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained from different times during the first six hours of MCMV infection revealed discrete functional networks of cellular genes regulated with distinct kinetics at surprising temporal resolution. Most of these were undetectable in total RNA, either due to low temporal sensitivity of standard gene expression profiling using total RNA, or due to rapid (viral) counter-regulation. Furthermore, metabolic labeling and purification of newly transcribed RNA detailed the real-time kinetics and regulation of viral gene expression in absence of any interfering virion-associated RNA. Both qRT-PCR and next-generation sequencing of newly transcribed RNA demonstrated an unexpected peak of viral gene expression during the first two hours of infection including transcription of immediate early, early and even well characterized late genes. Interestingly, this peak was subject to rapid gene silencing (independent of the multiplicity of infection) with similar transcriptional activity only reached late in infection. For some genes, e.g. m152, this resulted in massive down-regulation of transcriptional activity by 6 hours post infection, which was not even reversed late in infection. Our findings thus highlight the importance of transcriptional activity during the first few hours of CMV infection and hint at novel mechanisms governing the kinetics of viral gene expression. In conclusion, studying real-time transcriptional activity during lytic CMV infection provides intriguing new insights into the regulation of cellular and viral gene expression. In total 13 samples were examined: 3 samples of total RNA (mock, 25, 48 hours past MCMV infection), 3 samples pre-existing RNA (mock, 25, 48 hpi), and 7 samples newly-transcribed RNA (mock, 1-2, 5-6, 11-12, 18-19, 24-25, 47-48 hpi)

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Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis

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