Global Analysis of mRNA Half-Lives and de novo Transcription 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 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.

The Lingulodinium circadian system lacks rhythmic changes in transcript abundance

“…Indeed, these organisms do not contain detectable levels of histone proteins [ 28 ], their chromosomes are permanently condensed [ 29 ], and the number and diversity of transcription factors is also much reduced compared to other eukaryotes [ 16 , 30 ]. In addition, a global analysis of mRNAs in the dinoflagellate Karenia has shown their half-lives to be substantially longer than reported in other organisms [ 31 ], as might be expected if little control was exerted at the level of RNA synthesis rates. Lingulodinium also appears to have quite stable RNAs, as least for the few specific cases that have been examined [ 32 ].…”

Metatranscriptome profiling of a harmful algal bloom

“…At the level of mRNA presence and absence, these data suggest limited transcriptome re-modeling in P. minimum . This possibly reflects the highly duplicated nature of dinoflagellate genomes (Bachvaroff and Place, 2008; Beauchemin et al, 2012) and unusual transcriptional behavior (Morey and Van Dolah, 2013). The small number of gene ontology annotations that were significantly enriched in the environmental sample corresponds to core eukaryotic ontologies, such as ‘ribosomal component’ which had more than twice as many annotations in the environmental sample.…”

Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum

“…The observed changes in the Lhcf2 and Lhcf15 mRNA levels in response to DD could be regulated either at the level of transcription, mRNA stability or both. In vivo metabolic labeling of RNA with 4-thiouracil (4-TU) or 4-thiouridine is an efficient method to isolate labeled newly synthesized RNA fractions for determining mRNA synthesis and decay and has been developed in a range of eukaryotes 41,42 . We used 4-TU to label newly synthesized RNA because P. tricornutum contains the gene for uracil phosphoribosyltransferase (Uniprot ID B7FUW5), an enzyme for recycling uracil to uridine monophosphate in the pyrimidine salvage pathway 43 .…”