Exploring the interplay between mRNA degradation and ribosome dynamics using high-throughput sequencing

• Regulation of gene expression in response to fluctuating environments is essential for cellular survival. This regulation is multi-faceted, with mRNA abundance determined by both synthesis and decay. mRNA decay regulates transcript abundance, enabling swift transcriptomic adaptations. Several mechanisms, such as RNA-binding protein interactions and modulation of mRNA decay protein activity, regulate this decay in response to environmental changes. However, the mechanism linking mRNA decay with the translation process, known as co-translational mRNA decay, and its impact on mRNA stability is yet to be fully understood. In this thesis, we explore the intricate interplay between translation and mRNA decay, investigating its regulatory dynamics across varied physiological contexts and its role in cellular adaptations. In Paper I, we introduced a high-throughput 5'Pseq (HT-5Pseq) for a deeper exploration of the 5'P mRNA degradome in connection with translation. Our improved HT-5Pseq method is efficient, scalable, and cost-effective. This approach allowed us to investigate the significance of in vivo co-translational mRNA degradation footprints linked to ribosome stalling. In Paper II, we unexpectedly observed a massive ribosome protection pattern shifted back by 1 nt (- 1 nt) under extremely poor nutritional conditions using HT-5Pseq. We hypothesized that these -1 ribosome frameshifts accelerate out-of-frame co-translational mRNA decay. We characterized this mechanism and identified low codon optimality as a key factor prompting ribosomes to initiate outof- frame mRNA decay. We further established that this mechanism is conserved in both eukaryotes and prokaryotes. In Paper III, we demonstrated that codon optimality correlates with variations in mRNA stability of up to two-fold across various human tissues. This influence is less prominent in tissues characterized by high energy metabolism and becomes more accentuated with increased age. Using biochemical kinetic modeling, along with post-mortem samples from oxygen deprivation (using “Ischemic time” ) and HT-5Pseq with ATP synthesis perturbation using drugs, we confirmed that fluctuations in cellular energy differentially influence the decoding kinetics of various codons. In Paper IV, we investigated the regulations in transcriptional memory, an exemplary cellular mechanism for rapid adaptation to environmental changes. By performing a genome-wide screen in S. cerevisiae, we identified key contributors to transcriptional memory in response to galactose. We highlighted that depletion of the nuclear exosome component (RRP6) increased transcriptional memory. Furthermore, we showed how alterations in both nuclear and cytoplasmic mRNA decay processes influence transcriptional memory in primed cells.

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