Transfer RNAs (tRNAs) are the effector molecules of translation and are also a known source of small non-coding RNAs collectively known as tRNA-derived small RNAs (tsRNAs). tsRNAs have regulatory functions that range from translation regulation to gene expression control and cellular stress response, but what exactly triggers their formation is still under discussion. Both tRNAs and tsRNAs bear several modifications catalyzed by tRNA modifying enzymes. These modifications are essential for tRNA stability, translational efficiency and fidelity, and constitute the tRNA epitranscriptome. Although different enzymes, such as Dicer and Angiogenin (ANG), catalyze the formation of different classes of tsRNAs, the biological role of the tRNA epitranscriptome and whether it plays a role in tRNA fragmentation is only now beginning to be uncovered. Here, we describe how disruption of the 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs - one of the most common and conserved tRNA modifications among species - induces the generation of tsRNAs. Knockdown of the tRNA modifying enzyme TRMT2A in a human cell line induces m5U54 tRNA hypomodification, cellular stress and ANG up-regulation. The increase in ANG levels due to TRMT2A known-down results in tRNA cleavage near the anticodon, and accumulation of 5’tRNA-derived stress-induced RNAs (5’tiRNAs). Additionally, we demonstrate that exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNAs formation in mammalian cells. Our results unravel m5U54 as a tRNA cleavage protective mark, adding a further layer to the mechanisms associated with tsRNA generation upon stress. It also identifies TRMT2A as an important player in the cellular response to stress, and demonstrates that disruption of tRNA methylation is a trigger for tRNA fragmentation.

Transfer RNAs (tRNAs) are subjected to a wide variety of post-transcriptional modifications to ensure their structural stability, correct folding, and efficient protein decoding. More specifically, modifications in the D- and T-loops of tRNAs are essential for tRNA stability. However, the biological role of the tRNA epitranscriptome in the generation of tRNA-derived small RNA fragments (tsRNAs), a class of small non-coding RNAs, is still not totally understood. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. In mammals, this modification is catalyzed by the tRNA methyltransferase TRMT2A. To study the relevance of m5U54 for tRNA-derived small RNA formation, we have knockdown TRMT2A in human cells and found that m5U54 hypomodification resulted in ANG overexpression and tRNA cleavage near the anticodon, with accumulation of 5’tRNA-derived stress-induced RNAs (5’tiRNAs), in particular 5’tiRNA-GlyGCC and 5’tiRNA-GluCTC. Moreover, we found that exposure to oxidative stress induces TRMT2A downregulation, ANG overexpression and tsRNA generation. Our results establish a direct link between tRNA demethylation and ANG-dependent tRFs formation and propose the m5U54 as a tRNA cleavage protective mark.

Transfer RNA (tRNA) is the class of small RNAs with the highest number of chemical modifications. These modifications are catalyzed by multiple enzymes and ensure tRNA stability, as well as the efficiency and fidelity of mRNA translation, potentially leading to diseases known as tRNA modopathies. In recent years, significant progress has been made in understanding the biology of tRNA modifications; however, their impact on the proteome and human diseases is still not fully understood. In this thesis, we used yeast strains with deletions in genes encoding tRNA-modifying enzymes to further investigate the biology of these modifications. These strains were phenotypically and molecularly characterized and subjected to experimental evolution studies to assess their ability to adapt to tRNA hypomodification. The molecular mechanisms underlying this adaptation were also examined, and protein aggregation induced by tRNA hypomodification was monitored using a chimeric sensor (HSP104-GFP) that binds to protein aggregates. Strains deleted for the Elp1, Elp3, Trm4, Trm9, and Slm3 genes exhibited reduced growth and extensive protein aggregation. Surprisingly, they regained growth rates during laboratory evolution, despite maintaining protein aggregation at very high levels. Genome sequencing revealed the accumulation of copy number variations (CNVs), which disappeared over the course of evolution. RNA-seq data showed that all strains responded similarly to tRNA hypomodification, with a decrease in the expression of genes involved in stress response and protein folding, and an increase in the expression of genes related to protein synthesis at generation 500 in the strains deleted for Elp1, Elp3, and Trm9. The transcriptional response of strains deleted for Trm4 and Slm3 strains present the same response at the beginning of experimental evolution. Our data provides evidence that tRNA hypomodification results in protein aggregates accumulation, decreased fitness, gene expression and genomic alterations. The data also shows that such changes can be overcome over time, creating a homeostatic condition where protein aggregation is highly tolerated and does not affect fitness.

Viruses rely heavily on the host cell translation machinery, including host transfer RNAs (tRNAs), to efficiently translate their genomes. However, many viral RNA genomes, including the influenza A virus (IAV), are enriched in codons ending in adenine (A) or uridine (U), whereas the human genome is rich in cytosine (C) or guanine (G) ending codons. This is a challenge for viruses, as they require decoding of sub-optimal codons for which cognate host tRNAs are underrepresented. Nevertheless, despite the codon usage differences, codon-biased translation of IAV genes is highly efficient, suggesting the existence of viral strategies to manipulate host tRNA populations for optimal viral protein production. The reprogramming of host cell tRNA modifications upon infection may be one such strategy. tRNAs harbor a plethora of chemical modifications, collectively known as the tRNA epitranscriptome. These modifications are catalyzed by different classes of tRNA-modifying enzymes and, when they occur in the anticodon loop region, ensure the efficiency and fidelity of the translation process. These epitranscriptomic marks exhibit a dynamic behavior across cells and tissues, changing rapidly in response to environmental cues to maximize translation of stress-response genes. Given that viruses are sources of host cellular stress, it is possible that modulation of the hosts’ tRNA epitranscriptome occurs in this context. In this dissertation, I set to explore if and how host cell tRNA modifications were remodeled in a single cycle of IAV infection. The obtained results demonstrate that IAV infection affects the levels of mcm5U34 and mcm5s2U34 and of cognate tRNA modifying enzymes, including the elongator acetyltransferase complex subunit 3 (ELP3). To evaluate the relevance of ELP3 and its dependent modifications in the context of IAV infection, knockdown experiments were conducted. Loss of ELP3 induced tRNA hypomodifications and impaired translation of codon biased IAV genes. Moreover, the ELP3 knockdown triggered the integrated stress response (ISR) and interfered with the unfolded protein response (UPR)-related mechanisms, pivotal for IAV propagation. Specifically, silencing of ELP3 prior to IAV infection intensified the phosphorylation of the eukaryotic initiation factor 2 (eIF2α) and inhibited the inositol-requiring enzyme 1 α (IRE1α) axis concomitant to the accumulation of protein aggregates in the endoplasmic reticulum (ER) lumen of host-infected cells. Ultimately, induction of eIF2α likely amplified host antiviral responses linked to the NF-kB pathway, promoting the expression of interferon beta (IFN-β) and IFN regulatory factor 1 (IRF1). Taken together, our results uncover the relevance of the host cell tRNA epitranscriptome for optimal expression of viral genomes and host antiviral responses, setting the tRNA epitranscriptome as a promising host-based antiviral target.

Candida albicans is an opportunistic fungal pathogen responsible for most cases of invasive candidiasis. Normally a benign commensal of the human microbiota, C. albicans can transition to a pathogenic state under conditions that compromise host immunity or disrupt microbial balance. This shift relies on diverse virulence traits, including adherence, morphological transition, and biofilm formation. Recently, the epitranscriptome has emerged as an important regulatory layer of gene expression, virulence, and stress resistance of fungi. However, the elucidation of the role of the tRNA epitranscriptome in C. albicans pathogenicity remains poorly understood. In this study, six C. albicans clinical isolates from the Institute of Biomedicine (iBiMED) biobank, University of Aveiro, were analysed. Through APM-northern blotting we demonstrated that levels of the modification 2-thiolation at the wobble uridine of tRNA-LysUUU vary according to C. albicans strain. Moreover, we observed that the expression of two tRNA-modifying enzymes, NCS2 (involved in 2-thiolation) and HMA1 (that catalyses cyclic N6-threonylcarbamoyladenosine), were differently expressed across strains. Our results further correlate 2-thiolation levels with the formation of tRNA-derived fragments, and show that upon stress conditions, 2-thiolation levels are dynamically regulated, indicating that C. albicans relies on tRNA modifications as a putative means to adapt to stress conditions. Additionally, we characterised the clinical strains at the transcriptomic level by Illumina mRNA-sequencing and found that different strains depict altered levels of genes related to mRNA, rRNA and tRNA transcription and modifications. This provides important clues on how gene expression regulation can contribute to the infection and survival of the strains to different niches and virulence. Overall, these findings emphasize the importance of investigating tRNA modifications in C. albicans as this association could guide future development of novel antifungal therapies and clinical treatment strategies.