RESUMO
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.
Assuntos
Aminoácidos , Ritmo Circadiano , Dinoflagellida , Biossíntese de Proteínas , Transcrição Gênica , Aminoácidos/biossíntese , Aminoácidos/genética , Ritmo Circadiano/genética , Dinoflagellida/genética , Dinoflagellida/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
Dinoflagellates respond to daily changes in light and dark by changes in cellular metabolism, yet the mechanisms used are still unclear. For example, Fugacium (previously Symbiodinium) kawagutii shows little difference in the transcriptome between day and night suggesting little transcriptional control over gene expression. Here, we have performed ribosome profiling at 2 h intervals over a daily light-dark cycle to assess the degree to which protein synthesis rates might change over the daily cycle. The number of F. kawagutii coding sequences with significant differences in the number of ribosome-protected fragments (RPF) over the 24-h cycle was 2923 using JTK_Cycle and 3655 using ECHO. The majority of the regulated transcripts showed peak translation at the onset of the dark period. The regulated sequences were assigned to different KEGG pathways and transcripts that were translated at roughly the same time were termed concurrently regulated. Both analyses revealed concurrent regulation of many transcripts whose gene products were involved in spliceosome or lysosome biogenesis with peak translation rates around the onset of the dark period, while others, involved in nitrate metabolism and ribosomal proteins, were preferentially translated around the onset of the day phase or the end of the night phase, respectively. In addition, some sequences involved in DNA synthesis were preferentially translated at the end of the day. We conclude that light-dark cycles seem able to synchronize translation of some transcripts encoding proteins involved in a range of different cellular processes, and propose that these changes may help the cells adapt and alter their metabolism as a function of the time of day.
Assuntos
Dinoflagellida , Perfil de Ribossomos , Dinoflagellida/genética , Transcriptoma , Ribossomos/metabolismo , Regulação da Expressão Gênica , Perfilação da Expressão GênicaRESUMO
The dinoflagellate Lingulodinium specializes its metabolism to perform different tasks better at specific times of day. For example, cells are specialized for photosynthesis during the day and bioluminescence and cell division at night. These rhythms are circadian as they are controlled by an endogenous circadian clock whose mechanism is currently unknown. Despite this, the metabolic rhythms follow coordinated changes in gene expression that occur at a translational level. These changes are revealed by ribosome profiling, a surrogate measure of protein synthesis rates in vivo. Lingulodinium regulates the synthesis rate of over three thousand transcripts. Peak synthesis rates for the different transcripts are clustered around three different times over a light/dark cycle. Furthermore, transcripts involved in the same metabolic process are coordinately regulated. We review the basic principles underlying the correlation of coordinated translation of cell metabolic pathway enzymes with known circadian rhythms, and offer examples where previously unsuspected rhythms are suggested by synchronized changes in gene expression.
Assuntos
Relógios Circadianos , Dinoflagellida , Dinoflagellida/genética , Dinoflagellida/metabolismo , Perfil de Ribossomos , Ritmo Circadiano/genética , Biossíntese de ProteínasRESUMO
Protein levels were assessed in the dinoflagellate Lingulodinium polyedra over the course of a diurnal cycle using a label-free LC-MS/MS approach. Roughly 1700 proteins were quantitated in a triplicate dataset over a daily period, and 13 were found to show significant rhythmic changes. Included among the proteins found to be most abundant at night were the two bioluminescence proteins, luciferase and luciferin binding protein, as well as a proliferating cell nuclear protein involved in the nightly DNA replication. Aconitase and a pyrophosphate fructose-6-phosphate-1-phosphotransferase were also found to be more abundant at night, suggestive of an increased ability to generate ATP by glucose catabolism when photosynthesis does not occur. Among the proteins more abundant during the day were found a 2-epi-5-epi-valiolone synthase, potentially involved in synthesis of mycosporin-like amino acids that can act as a "microbial sunscreen", and an enzyme synthesizing vitamin B6 which is known to protect against oxidative stress. A lactate oxidoreductase was also found to be more abundant during the day, perhaps to counteract the pH changes due to carbon fixation by facilitating conversion of pyruvate to lactate. This unbiased proteomic approach reveals novel insights into the daily metabolic changes of this dinoflagellate. Furthermore, the observation that only a limited number of proteins vary support a model where metabolic flux through pathways can be controlled by variations in a select few, possibly rate limiting, steps. Data are available via ProteomeXchange with identifier PXD006994.