Comprehensive analysis of the circadian nuclear and cytoplasmic transcriptome in mouse liver

In eukaryotes, RNA is synthesised in the nucleus, spliced, and exported to the cytoplasm where it is translated and finally degraded. Any of these steps could be subject to temporal regulation during the circadian cycle, resulting in daily fluctuations of RNA accumulation and affecting the distribution of transcripts in different subcellular compartments. Our study analysed the nuclear and cytoplasmic, poly(A) and total transcriptomes of mouse livers collected over the course of a day. These data provide a genome-wide temporal inventory of enrichment in subcellular RNA, and revealed specific signatures of splicing, nuclear export and cytoplasmic mRNA stability related to transcript and gene lengths. Combined with a mathematical model describing rhythmic RNA profiles, we could test the rhythmicity of export rates and cytoplasmic degradation rates of approximately 1400 genes. With nuclear export times usually much shorter than cytoplasmic half-lives, we found that nuclear export contributes to the modulation and generation of rhythmic profiles of 10% of the cycling nuclear mRNAs. This study contributes to a better understanding of the dynamic regulation of the transcriptome during the day-night cycle. Author summary Why was this study done? The circadian clock coordinates temporal gene expression programs to synchronise cellular and physiological functions with environmental time.The circadian clock impinges on many layers of gene regulation, from transcription to mRNA degradation in the cytoplasm.Specifically, whether temporal modulation of nuclear export could influence rhythmic mRNA expression was not known. What did the researchers do and find? We sequenced RNA from nuclear and cytoplasmic fractions from mouse livers sampled at different times of the day.Using a mathematical model describing temporal accumulation profiles of pre-mRNA, nuclear mRNA and cytoplasmic mRNA, we could estimate the nuclear export rates and cytoplasmic degradation rates of approximately 1400 genes.We observed rhythmic patterns in the nucleus that most likely originate from a rhythmic regulation of the nuclear export rate, affecting approximately 10% of the oscillations of the nuclear transcriptome in the liver. What do these findings mean? Our fractionated RNA analysis contributes to a better understanding of the dynamic regulation of the transcriptome during the daily cycle.The majority of the RNA transcripts are exported to the cytoplasm in less than 30 minutes, however, some are retained in the nucleus for several hours. Thus, cellular fractionation allows a finer resolution of temporal profiles in the different subcellular compartment, which is otherwise masked at bulk-level.

Circadian dynamics of RNA localisation in the mammalian liver

Clémence Yumie Syloun Hurni

Gene expression in eukaryotes is a complex multi-step process. It starts in the nucleus with transcription, the synthesis of a mRNA copy from a DNA template. While in the nucleus, the RNA transcript is subject to multiple co- and post-transcriptional modifications, including splicing, capping, polyadenylation, and assembly into ribonucleoprotein complexes. Correctly processed mRNA is exported to the cytoplasm and translated by ribosomes to form a chain of amino acids: the protein. The mRNA lifetime in the cytoplasm is determined by the activity of a distinct set of RNA Binding Proteins, enzymes, and functional RNAs, which promote stability or degradation. Each step of the RNA life cycle is tightly regulated to ensure proper cellular function. The kinetic rates governing these steps are dynamic and notably adapt to the daily fluctuations of the environment related to the alternance of day and night. Indeed, most organisms possess an internal timing system called the circadian clock. The clock is a genetically encoded self-sustained transcriptional-translational feedback loop, which operates in almost every cell and tissue of the body. It controls the temporal gene expression program to synchronise cellular and physiological functions to the external world. In this thesis, I explore the transcriptome of the mouse liver by combining RNA-sequencing, mathematical modelling, and single-molecule RNA-FISH (smFISH), with an emphasis on the spatio-temporal organisation of RNA expression at the subcellular and tissue scales. I first investigate how RNA are differentially localised at the scale of the liver tissue. Hepatocytes are arranged in structural and functional units called lobules, and carry out different physiological functions depending on their spatial position within the lobule. In this collaborative work, we characterised spatio-temporal gene expression profiles, and showed that that while the expression of hundreds of genes is dually orchestrated by time and space, the circadian core clock is expressed uniformly within the liver lobule, and is therefore robust to the heterogeneous microenvironment. Second, I explore the RNA localisation at the scale of a hepatocyte. The subcellular distributions of RNA in different compartments (here, the nucleus and the cytoplasm), are dictated by the balance of a synthesis term and a decay term. To quantify the kinetic parameters driving nuclear and cytoplasmic mRNA accumulation, I sequenced RNA from both cellular fractions from mouse livers sampled at different times of the day. Using a mathematical model describing rhythmic pre-mRNA and mRNA profiles, I could estimate the nuclear export rates and cytoplasmic degradation rates of

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1400 genes. Nuclear export occurs on a much shorter time-scale than cytoplasmic degradation, and nuclear lifetime has only a minor contribution to the total RNA lifetime. However, a subset of metabolic genes remain in the nucleus for more than one hour (up to four hours), which accounts for the long phase delay between the peak times of transcription and of cytoplasmic accumulation. Furthermore, nuclear export contributes to the modulation and generation of rhythmic profiles of ~10% of the cycling nuclear mRNA. This study provides a comprehensive estimation of the nuclear and cytoplasmic life times in the liver and contributes to a better understanding of the dynamic regulation of the transcriptome during the feeding-fasting cycle.

EPFL2021

Kinetic Analysis of Transcriptional and Post-Transcriptional Processes During Circadian Cycles

Laura Symul

Every morning the sun rises, flooding the earth with its light and heat and every night it disappears leaving the Earth in the darkness of the night. Organisms living on this planet have adapted to this solar rhythm and have spread their activities throughout the day. Early in the development of life, an internal molecular clock appeared in primitive organisms and the evolutionary advantage of being able to anticipate, rather than simply passively face, environmental changes led virtually all organisms to have an internal timing system called "circadian clock." In most species, this clock is constituted of a genetic network ticking in every cell of the body. This genetic network organized in interlocked feedback loops generates oscillations with a 24-hour period. A variable number of proteins and RNAs is thus accumulating in a rhythmic fashion. For example, in mouse liver, which was the main biological model I used during my thesis, it is estimated that thousands of genes are expressed periodically. The molecular processes leading to rhythmic gene expression and protein accumulation are highly regulated. Dynamical control is needed to sustain the oscillations and to maintain their timing. During my PhD, I was particularly interested in the regulation of rhythmic gene activation via transcription factor binding in their promoter region. I also focused on gene transcription itself and its dynamics as well as on transcription associated changes in epigenetic marks. Another interest was given to the regulation of transcripts (messenger RNA) after transcription, especially to understand whether their degradation in the cytoplasm was regulated rhythmically. Finally, I contributed to a project aiming at the detection of transcripts whose translation into protein was specifically regulated in a circadian manner. The four projects on which I worked were done in collaborations with experimental biology labs. Our partners performed the experiments and provided the biological material while I was in charge of the data processing and their statistical analysis. For some projects, I also developed different mathematical models for a more accurate and complete understanding of the phenomena studied and to take into account the inherent variability of biological data. Notably in the project on the post-transcriptional regulation of transcripts, the model-based approach we developed allowed estimating the role of this regulation in a way that is less prone to arbitrary choice of criteria or thresholds compared to previous studies addressing this question. To summarize, the results I obtained show that, in human cell lines, the major transcription factor of the clock (the heterodimer CLOCK/BMAL1) binds to DNA at specific binding sites and their number is comparable to the number of binding sites observed in the mouse liver. However the fraction of rhythmic transcript among the genes with CLOCK/BMAL1 binding sites is about 10 times lower than the corresponding fraction observed in mouse liver. The results of the second project showed that, in mouse liver, rhythmic transcription is followed by dynamical changes in epigenetic marks with delays specific to the marks (tri-methylation of lysines 4 and 36 of histone 3 of nucleosomes) and we highlighted the role of post-transcriptional regulation for the rhythmic expression of a subset of mRNAs. In the third project, we further investigated the regulation at the post-transcriptional level and combining measurements of pre-mRNA and mRNA with a mathematical model, we showed that the post-transcriptional regulation plays a limited role in the rhythmic accumulation of transcripts and is involved in the regulation of only 20 % of rhythmic transcripts. Laboratories of Michael Brunner (University of Heidelberg, Germany), Frederic Gachon, Nouria Hernandez (University of Lausanne, Switzerland) and Ueli Schibler (University of Geneva, Switzerland) 3 Finally, helped by the analysis I performed on micro array data from an experiment aiming at comparing the fraction of translated mRNA to the total accumulation of these mRNAs, the team of Frederic Gachon showed that several transcripts implied in the composition of ribosomes are translated specifically during the night, when rodents are active. All of these results and their links with other published studies on circadian rhythms, show that biological tissues have largely incorporated the clock for the regulation of many other functional pathways in order to optimally synchronize their physiological actions with environmental changes due to the alternation of days and nights. In mouse liver, links with the metabolism are particularly strong and disruptions of the clock induce phenotypes reflecting a disturbed metabolism.

EPFL2013