The crosstalk between miRNAs and tRNA modifying enzymes: an alternative pathway to regulate the tRNA epitranscriptome

• Autor(a) principal: Bastos, Diana Gisela Silva Barbosa
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/35611
• topic: tRNA modifications; tRNAs; Proteostasis; MicroRNAs; Gene expression regulation

Non-coding RNAs, namely transfer RNAs (tRNAs) and microRNAs (miRNAs) are pivotal for accurate translation of mRNAs into proteins. tRNAs, the adaptor molecules of translation, carry several chemical modifications (tRNAMods). These are catalysed by tRNA modifying enzymes (tRME) and are crucial for translation accuracy and fidelity. Indeed, imbalances in tRNAMods and in tRME expression are found in cancer and neurological disorders. Although both imbalances in tRNAMods and in miRNA expression have been pinpointed as causes of translation impairments and pathogenesis there is a lack of studies exploring how and if miRNAs are recruited in response to tRNA hypomodification. Since miRNAs control gene expression post-transcriptionally, we hypothesize that impaired translation efficiency due to tRNAMods disruption leads to cellular translation reprograming through miRNA-regulated mechanisms. Taking advantage of preliminary data from the host group on -omics analysis of HeLA cells silenced for a specific tRME – ELP3, as well as sncRNA-Seq data in the same cell line, I started by performing data integration analysis to identify miRNA candidates that could play a role in the cellular response to tRNA hypomodification. This analysis revealed that ELP3 silencing led to decreased abundance of other tRME – TRMU, and that both enzymes were putative miR-1-3p targets. To experimentally validate these findings, ELP3 and TRMU expression was challenged and their expression levels, as well as miR-1-3p levels, were quantified. Additionally, cells were transfected with miR-1-3p mimics and miRNA inhibitors and ELP3 and TRMU mRNA and protein expression was assessed, as well as other relevant factors, namely protein aggregation levels, and the unfolded protein response. Binding of miR-1-3p to the 3’UTR of ELP3 was tested and validated with Dual reporter Luciferase assays. This thesis shows that tRNAME enzyme disruption, and consequently tRNAMod imbalances, influence miRNA expression and that, in turn, miRNA dysregulation impacts tRNAME expression. These results provide the first evidences of a crosstalk between miRNAs and tRNA epitranscriptome modulation, demonstrating that miRNAs can also be used to predict tRNA modification levels and may represent promising targets to promote tRNA modification reprograming in conditions where those epitranscriptomic marks are affected, namely conformational disorders or cancer.