The Lingulodinium circadian system lacks rhythmic changes in transcript abundance

Abstract

BackgroundAlmost all cells display circadian rhythms, approximately 24-hour period changes in their biochemistry, physiology or behavior. These rhythms are orchestrated by an endogenous circadian clock whose mechanism is based on transcription-translation feedback loops (TTFL) where the translated products of clock genes act to inhibit their own transcription.ResultsWe have used RNA-Seq to measure the abundance of all transcripts in an RNA-Seq-derived de novo gene catalog in two different experiments. One compared midday and midnight in a light–dark cycle (ZT6 and ZT18) and under constant light (CT6 and CT18). The second compared four different times (ZT2, ZT6, ZT14 and ZT18) under a light dark cycle. We show here that despite an elaborate repertoire of biological rhythms, the unicellular dinoflagellate Lingulodinium had no detectable daily variation in the abundance of any transcript in an RNA-Seq-derived de novo gene catalog. We also examined the timing of the bioluminescence and photosynthesis rhythms in the presence of the transcription inhibitors actinomycin D and cordycepin. We found that the timing of the two rhythms was unchanged even when transcription rates had decreased to roughly 5% the levels of untreated cells.ConclusionsThe lack of detectable daily variation in transcript levels indicates that the endogenous circadian timer of Lingulodinium does not require rhythmic RNA. If the circadian timer is considered as a limit cycle oscillator, then cellular time in this organism must be defined by variations in state variables that do not include the amount of a clock gene transcript.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0107-z) contains supplementary material, which is available to authorized users.

miRNAs Do Not Regulate Circadian Protein Synthesis in the Dinoflagellate Lingulodinium polyedrum

“…polyedrum , a species where the circadian clock regulates gene expression post-transcriptionally [6]. Northern blots using a highly expressed miRNA sequence from S .…”

“…The number of scintillons [3], as well as the cellular levels of both luciferase [4] and LBP [5] all correlate with the bioluminescence capacity of the cells. The regulation of both luciferase and LBP synthesis occurs at a translational level since mRNA levels for both are constant [5, 6]. The mechanism for this is still unclear, however, as there are conflicting reports concerning the action of an inhibitory protein binding to cis-acting sequences in lbp RNA during the day phase [7, 8].…”

Refining Transcriptome Gene Catalogs by MS‐Validation of Expressed Proteins

“…We were also curious about the degree to which RNA and protein levels were correlated in the dinoflagellate Lingulodinium , as this species has daily changes in protein synthesis rates but does not alter RNA levels to accomplish this . We thus predicted that the correlation between the two might be on the low side compared to what has been observed in other systems.…”

“…Protein levels are estimated from ion intensities; when more than three peptides are present for a single protein, protein levels are determined using the three most abundant peptide ion intensities. Correlations to RNA levels determined previously using RNA Seq data were evaluated using the ggpubr package in R.…”

Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

“…The transcriptional rhythms in Nannochloropsis species could also be controlled by a damped oscillator. The dinoflagelate Lingulodium , a model of early circadian studies due to its robust endogenous bioluminescece rhythms, does not appear to have strong RNA oscillations under constant light conditions (Roy et al , 2014) although it does display rhythms in protein synthesis (Morse et al , 1989, Morse et al , 1990). Future studies will reveal the role of posttranscriptional regulation of the circadian rhythms in the Nannochloropsis genus.…”