Circadian rhythms allow organisms to anticipate and adapt to periodic environmental changes related to the day-night cycle. These rhythms originate from a genetic network ticking in almost every cell of our body and the coordination between different organs, thus allowing to generate physiological outputs at the right time of the day. In the liver, which is one of the major metabolic organ of the body, gene expression has to be highly regulated by the circadian clock and feeding rhythms to generate such a timely behavior. Indeed, at every step from transcription to post-translational modifications, the circadian clock and metabolism interplay.
In this context, we quantified transcription, mRNA accumulation and translation in liver from wild-type and clock-deficient mice fed ad libitum or only during the night period. RNA-Sequencing and ribosome profiling combined with mathematical modeling were used to assess rhythmic regulation along the gene expression process. Transcription was found to be the main driver of temporal mRNA accumulation and its subsequent translation. We observed a diurnal translation efficiency in genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences or with Translation Initiator of Short 5'-UTR (TISU) motifs. Remarkably, the rhythmic translation efficiency was mainly driven by feeding cues although clock-deficiency slightly affected the amplitude and phase of the oscillations.
In a subsequent work, we emphasized the above analysis of translation regulation with a focus on the ribosome elongation process. A statistical model and bioinformatics pipeline were developed to infer codon dwell times from ribosome profiling data. The method has the particularity of integrating pairs of codons between the sites of the ribosome and to fit every sequencing read on an individual basis. We validated our approach on a yeast dataset and then studied extensively the mouse liver translation elongation landscape. A large dynamic range between the fastest and longest codons was uncovered in the ribosome A and P sites with strong synergic interaction of pairs of codons clustering by amino acids. In order to challenge our system, we performed drug free ribosome profiling, modified to reduce library biases, in liver of mice fed ad libitum or starved. Unexpectedly, codon dwell times were conserved between the two conditions despite a gene expression landscape reflecting low energy state of the mice and subsequent amino acid shortage.
Finally, to understand how the ribosome elongation velocity is regulated by (aminoacyl)-tRNA levels, we adapted and modified a published DNA hybridization-based tRNA profiling method in liver of mice fed ad libitum or starved. While the quantified tRNAs abundance showed a large dynamic range and specific isoacceptors patterns, no differences in (aminoacyl)-tRNAs were found between the two feeding conditions. Nevertheless, tRNAs for isoleucine, asparagine, aspartate and arginine exhibited a relative low loading state compared to other tRNA in both conditions. Altogether, correlation between codon usage, codon dwell times and tRNA abundances provided insights of how translation elongation is regulated in mammals. Together, this thesis aimed to dissect the mechanisms regulating gene expression by the interplay of the circadian clock and metabolism, as well as understand how translation elongation is regulated in mammals.
EPFL2018
