Visualising autoregulation: the development of a fluorescent reporter

Diplôme Master Visualising autoregulation: the development of a fluorescent reporter eng 2024-06 author Fischer Olivia Lucie 859779 director Gasic Ivana 0000-0002-4824-8174 796323 Département de biologie moléculaire et cellulaire (MOCEL) 73 <p>Tubulin autoregulation is a post-transcriptional and co-translational regulation of tubulin triggered by a sudden increase in soluble tubulin concentration. The recognition of the tubulin nascent chain exiting the ribosome tunnel leads to the deadenylation of tubulin mRNA and its subsequent degradation. Despite recent advances, many questions remain unanswered: the regulation of the pathway and its physiological function in cell processes, among others. This study presents the initial steps in the design and optimisation of a fluorescent reporter – a novel in vivo light microscopy tool to visualise tubulin autoregulation. The reporter consists of the autoregulation motif – the first four amino acids of tubulin and essential to the recognition of tubulin as a target for mRNA degradation – fused to a fluorescent protein, a protein-degradation sequence and a nuclear localisation signal (NLS). The aim is to harness the autoregulation pathway to degrade reporter mRNA as well as observing a time dependent fluorescence decrease, reporting a functional pathway. Our attempts at generating and optimising a functional reporter proved unfruitful, as the several iterations of constructs were not affected by the activation of the pathway, but also interfered with autoregulation of the endogenous tubulin expression. Our analysis suggests that the overexpression of the reporter might be too efficient. The high abundance of reporter mRNA might saturate some factors of the pathway. The structure of the reporter may also cause the forced localisation to the nucleus of an essential factor of the tubulin autoregulation pathway, TTC5 (tetracopeptide protein 5). We propose that the reporter interacts with TTC5 through its N-terminal and localises to the nucleus due to its NLS. This may lead to the inability of TTC5 to fulfil its role in the tubulin autoregulation pathway. This study highlights important aspects to be taken into consideration for future design of a functional reporter to monitor tubulin autoregulation.</p> Tubulin Microtubules Tubulin autoregulation Post-transcriptional regulation MRNA Fluorescent reporter RT-qPCR High-throughput microscopy Immunofluorescence Microtubule drugs Dynamic instability Dewey 570 Master in Biology, option Genetics, Development and Evolution 7daed2ee-1311-4824-9df9-2e51260a966a Département de biologie moléculaire et cellulaire 16484 false 1359 Groupe Gasic 15009a44-2407-44e5-aa89-ad7de94e5986 Master thesis Restricted Master_thesis_230624-final.pdf 2630887 application/pdf 4cd048d3024ed84b1e2ac0bcbe169b4ee5be717069fce7e4b246c94615b179a9 restrictedAccess Faculté des sciences / Section de biologie / Département de biologie moléculaire et cellulaire

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Visualising autoregulation: the development of a fluorescent reporter				2024	66	5

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Fischer, Olivia

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