Dinoflagellates are an important component of the marine biota, but a large genome with high–copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

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.

Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists.

Dinoflagellate chromatin is unique among eukaryotes, as the chromosomes are permanently condensed in a liquid crystal state instead of being packed in nucleosomes. However, how it is organized is still an unsolved mystery, in part due to the lack of a comprehensive catalog of dinoflagellate nuclear proteins. Here, we report the results of CHromatin Enrichment for Proteomics (CHEP) followed by shotgun mass spectrometry sequencing of the chromatin-associated proteins from the dinoflagellate Lingulodinum polyedra. Our analysis identified proteins involved in DNA replication and repair, transcription, and mRNA splicing, and showed a low level of contamination by proteins from other organelles. A limited number of proteins containing DNA-binding domains were found, consistent with the lack of diversity of these proteins in dinoflagellate transcriptomes. However, the number of proteins containing RNA-binding domains was unexpectedly high supporting a potential role for this type of protein in mediating gene expression and chromatin organization. We also identified a number of proteins involved in chromosome condensation and cell cycle progression as well as a single histone protein (H4). Our results provide the first detailed look at the nuclear proteins associated with the unusual chromatin structure of dinoflagellate nuclei and provide important insights into the biochemical basis of its structure and function.

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.