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Global Analysis of mRNA Half-lives 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 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.

ORGANISM(S): Karenia brevis

PROVIDER: GSE46174 | GEO | 2013/08/01

SECONDARY ACCESSION(S): PRJNA197496

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 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.

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Global Analysis of mRNA Half-lives Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis

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