microRNA are short non-coding RNAs and important post transcriptional gene regulators. miRNAs are found in all animals that have been studied, in numbers that largely correlate with organismal complexity. For instance, nematodes have around 200 miRNA genes, while humans have more than 600 miRNA genes. Mutant animals that are deficient in miRNAs generally exhibit gross developmental defects or embryonic lethality, underlining the importance of these regulators. Given that an estimated ~60% of human mRNAs are targeted by miRNAs in some cellular context, it is not surprising that these regulators are involved in numerous biological processes, ranging from the formation of cell identity to development and human diseases. Even though miRNAs have been systematically studied for over a decade, fundamental questions regarding their biogenesis and function remain unanswered. microRNAs are unevenly distributed between cell types and within homogeneous cell populations, affecting the transcriptomes of individual cells. The vast majority of miRNA studies have been conducted on large pools of cells, and little is known about the biogenesis and function in individual cells. To understand their effect on gene regulation, single-cell measurements are crucial. This thesis introduces techniques that allow us to extend our understanding about microRNA at the resolution of single cells.

In Paper I, we develop and establish agoTRIBE – the first sequencing-based method to measure regulatory interactions between miRNAs and their mRNA targets transcriptome-wide in single cells. We applied Smart-seq3 single-cell RNA sequencing to detect increased editing transcriptome-wide in key miRNA targets and found substantial differential targeting across the cell cycle and in mixed cell populations. This method overcame limitations of current methods and allowed for study of heterogeneity in miRNA targeting across individual cells. In Paper II, we further explored miRNA targeting landscape in single cells using agoTRIBE and revealed differential targeting within homogenous cell populations. We observe two groups of cells with overlapping but distinct targeting patterns and provide evidence that miRNAs act on different groups of genes with distinct biological functions. In paper III, we proposed a method 'micro-imp' to infer miRNA activity from existing single cell RNA-sequencing (scRNA-seq) data, to overcome the limitation of low sensitivity in direct miRNA sequence of single cells. We show significant positive correlation between the inferred activity and the measured miRNA expression suggesting that this approach can be utilised to obtain orthogonal information from existing scRNA-seq datasets. In paper IV, we provide valuable insights from miRNA profiling in single cells and together with integration of scRNA-seq data from the same study system. We highlight heterogeneity in miRNA expression across single cells and link it to the variation and covariation of the target pool. Further, we detect large parts of pri-miRNA transcripts in single cells devoid of biogenesis factors, an aspect previously underexplored. The work presented in this thesis focus on methods and techniques to expand our understanding of miRNA biology at single cell resolution.