RNA metabolism as a readout of cellular fitness and antimicrobial resistance

<jats:p>&lt;p dir="ltr"&gt;Detecting antimicrobial resistance using standard microbiology assays relies on measuring cell growth (a phenotype) that requires days. Molecular methods based on qPCR are faster but require prior knowledge of the resistance mechanisms, thus are limited in scope. To fill this gap, we investigated whether translation measurements and RNA decay could be exploited to obtain a fast molecular readout of cellular stress and predict antimicrobial drug effects.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper I, we investigated whether ribosome dynamics associated to mRNA decay provide a readout of cellular stress that does not directly interfere with translation. Using 5' monophosphorylated RNA sequencing (5PSeq), we characterized the response to cell membrane stress induced by fluconazole in fluconazole-sensitive and resistant Candida albicans strains. First, we established that co-translation decay occurs in C. albicans and validated the 5PSeq method to study ribosome dynamics at codon resolution by perturbing translation (though depletion of specific amino acids). We then examined ribosome protection associated to 5-3' mRNA decay and observed a global decrease in translation frame protection, but this effect was present in both sensitive and resistant strains. Finally, we characterized the early transcriptional changes in these strains and identified distinct transcriptional programs even in the absence of treatment, likely reflecting intrinsic genetic determinants of resistance in C. albicans.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper II, we built 5PSeq Explorer, a web platform aggregating multiple stresses (antibiotics, osmotic stress, heat stress) with associated ribosome dynamics from 5PSeq experiments. We established a uniformly processed degradome database suitable for downstream analyses, and a web interface for visual exploration of translation measurements. Using the accumulated data, we found that codon specific ribosome pauses, interpreted in the context of mRNA stability, provide a valid readout of stress targeting translation.&lt;/p&gt;&lt;p dir="ltr"&gt;Taken together, the studies in this thesis establish a foundation of ribosome signatures associated to cellular stress for future use of 5' monophosphorylated RNA sequencing (5PSeq) applications.&lt;/p&gt;&lt;h3 dir="ltr"&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;I. &lt;b&gt;Stevens I,&lt;/b&gt; Silao, F. G., Huch, S., Liu, H., Ryman, K., Carvajal-Jimenez, A., Ljungdahl, P. O., &amp; Pelechano, V. (2024). The early transcriptional and post-transcriptional responses to fluconazole in sensitive and resistant Candida albicans. Scientific reports, 14(1), 29012. &lt;a href="https://doi.org/10.1038/s41598-024-80435-w" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1038/s41598-024-80435-w&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. &lt;b&gt;Stevens I,&lt;/b&gt; Pelechano, V. (2026). 5PSeq Explorer: interactive analysis of co-translational mRNA decay and ribosome dynamics. RNA Biology, 23(1), 1-10. &lt;a href="https://doi.org/10.1080/15476286.2026.2639616" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1080/15476286.2026.2639616&lt;/a&gt;&lt;/p&gt;</jats:p>