Gene expression analysis during aging of the annual fish Nothobranchius furzeri

The broad topic of this thesis is aging, that was studied through the analysis of transcriptomic and miRNomic data with the aim of finding molecular features and potential regulators conserved across species. A specific focus was set on brain and mechanisms causing neurogenesis decline. Base of the study was Next Generation Sequencing (NGS) JenAge dataset (cf. 1.5.1), comprising several species (C. elegans, N. furzeri, D rerio, M. musculus) and tissues (brain, liver, blood, skin) at different time points during aging (at least 5 ages with 5 replicates for each organism). The work was developed on three lines: transcriptome, miRNome and IsomiR analysis, often combined and converging in the investigation of causes and consequences of microRNAs regulation. Transcriptome was studied in N. furzeri brain, where, expanding a previous work (Baumgart et al., submitted), Gene Ontology enrichment analysis on differentially expressed genes and correlation networks led to the identification of several genes regulated with age and specifically expressed in neurogenic niches. Among others was the uncharacterized zinc finger ZNF367, central hub of the network, which can thus be considered an interesting candidate for further studies in neurogenesis regulation. MiRNome was analysed starting from raw NGS data that were hence quantified and annotated to find evolutionarily conserved differentially expressed microRNAs in the brain. Independently, microRNAs potentially regulating gene expression in N. furzeri brain were identified comparing microRNAs and their predicted targets’ expression profiles. These analyses converged on two microRNAs, miR-29a and miR-101a, previously shown to be onco-suppressors. These microRNAs were assayed by in situ hybridization in N. furzeri brain cryosection, revealing their specific expression in differentiated neurons, and overexpression experiments via injections into zebrafish embryos, showing their role in cell cycle escape. Finally, they were shown to alter the transcriptome (in overexpression experiments) in a way that resembles physiological aging. IsomiR analysis revealed the existence of a remarkable variability in microRNA sequences, which were studied in their general features (position along the precursor, length, mismatches number, type and position) and in their variation with age. Specifically, microRNAs were found being shortened with age via 3’ end trimming in the brain of all the analysed organisms and being subject to an increase in mismatches at their 3’ end in all tissues and organisms. Moreover, several internally edited forms, differentially expressed with age, were identified and studied more deeply in mouse brain where they were implicated in neurogenesis and plasticity decline. Surprisingly, loops deriving from microRNA precursor hairpins were discovered being negatively correlated with their predicted targets and hence potentially physiologically active in gene expression regulation. A few enzymes potentially causing microRNA sequence variability (Nibbler, Hen1 and ADAR – Adenosine Deaminase Acting on RNA) were studied and shown to be probably implicated in the observed features and their change with age. In conclusion, this thesis led to identification of several regulators of aging, such as ZNF367, miR-29a and miR-101a, and molecular mechanisms of aging, such as microRNA shortening and editing, worth further investigations