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 roles of differential mRNA stability and de novo transcription 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 and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to 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 144 h, with a median of 33 hours. 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. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase 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 increases 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.
Highlights
Phytoplankton are essential primary producers, responsible for up to 70% of the world’s oxygen production
In many eukaryotes, uridine kinase (UK) has higher activity than uracil phosphoribosyltransferase (UPRT) leading to more efficient salvage of uridine rather than uracil [41]
UK-UPRT fusion genes encoding bifunctional proteins with both UK and UPRT activities are found across eukaryotic the tree of life, but are notably absent from the nearest neighbors to dinoflagellates, the apicomplexans, except for Cryptosporidium parvum, and have not previously been identified in dinoflagellates [42]
Summary
Phytoplankton are essential primary producers, responsible for up to 70% of the world’s oxygen production. As HABs have significant ecological, human health, and economic impacts [1] much recent attention has been focused on their molecular biology to better understand the mechanisms underlying bloom dynamics and toxicity for improved coastal management and forecasting. These unicellular protists have several unusual nuclear traits, including exceptionally large genomes of 3–2456106 kbp [2]. In Karenia brevis cell cycle genes, typically under transcriptional control in most organisms, appear to be post-transcriptionally regulated [15], [16]. Microarray and MPSS cannot determine what mechanism(s) are responsible for the measured changes in transcript abundance
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