Non-coding RNAs (ncRNAs) play essential roles in gene regulation, cellular function, and evolution. They act as key regulatory elements in diverse biological processes, such as influencing transcription, splicing, post-transcriptional regulation, and host-pathogen interactions. Despite significant advancements in ncRNA research, fundamental questions remain regarding their regulatory mechanisms and functional impact across different biological contexts. This thesis explores various aspects of ncRNA biology by integrating bulk and single-cell transcriptomics, evolutionary analyses, and infection biology to provide deeper insights into their roles in gene regulation, evolution, and diseases.

In Study I, we investigate the gene regulatory roles of Malat1, a highly expressed long non-coding RNA, in mouse embryonic stem cells. By employing knock-down and bulk RNA sequencing, we identify genes and pathways regulated by Malat1 at both the transcriptional and post-transcriptional levels, shedding light on possible roles in stem cell maintenance and differentiation.

In Study II, we explore the evolutionary landscape of microRNAs by analyzing their structural and functional features across 114 metazoan species. Using MirGeneDB 3.0, we identify conserved and lineage-specific miRNA characteristics, revealing how evolutionary pressures have shaped miRNA expression, processing efficiency, and regulatory function over millions of years.

In Study III, we evaluate the predictive power of single-cell RNA sequencing for gene regulatory network inference. By comparing RNA-based and protein-based regulatory predictions in single cells to gold standard datasets - bulk RNA-seq data for expression analysis and ChIP-seq data for direct transcription factor binding - we assess the reliability of single-cell approaches for reconstructing regulatory interactions and discuss key limitations of single-cell-based inference.

In Study IV, we examine the impact of Toxoplasma gondii infection on neuronal miRNA profiles. Through the analysis of extracellular vesicle-associated miRNAs, we identify infection-induced changes that may contribute to host-pathogen interactions and neuronal cytoskeletal remodeling.

This thesis provides a comprehensive framework for understanding ncRNA functions across multiple biological contexts, integrating perspectives from stem cell biology, gene regulation, evolutionary analysis, and infection biology. The findings contribute to fundamental questions in ncRNA research, offering insights into how non-coding RNAs shape cellular identity and disease mechanisms.