Circadian dynamics of RNA localisation in the mammalian liver

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 $\sim$ 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.