The advent of high-throughput RNA sequencing has enabled, for the first time, direct and global investigation of the vast complexity of eukaryotic transcription. One of the main findings of these transcriptomic surveys was the existence of a variety of small RNAs that are specifically expressed in particular environmental or developmental contexts, which are capable of acting as master regulators of specific biological pathways. There are now at least ten established small RNA classes, some of which have been deeply and functionally characterized, and include miRNAs, piRNAs, siRNAs, among many others. Due to the small size of these transcripts (between 16 to 30 nts long) it is believed the majority of these small RNAs regulate other longer and partially complementary RNA transcripts. During my thesis, I have made use of this revolutionary technology to characterize novel biological functions and features of miRNAs, as well as to discover new classes of small RNAs.
In Chapter 1 I provide the reader with an introduction to the RNA world, focusing on the classes and functions of small RNAs characterized thus far.
Chapter 2 presents a comprehensive analysis of differential microRNA expression in gonad differen- tiation, in which I identified a small cohort of miRNAs differentially expressed between males and females during gonad differentiation. Indeed, in this chapter I show that miR-140-3p is highly upreg- ulated in the developing testis, and that lack of this miRNA corresponds to an increase in a gonadal cell type involved in fetus masculinization.
Next, in chapter 3, I present the first study to show that particular microRNA isoforms, called isomiRs, are differentially regulated during Drosophila melanogaster development. This landmark study helped solidify the biological relevance of isomiRs, which had previously been disregarded as sequencing artefacts.
Chapter 4 extends the work presented in Chapter 3 and shows that isomiRs are present and differen- tially regulated in a panel of ten human tissues. In addition to validation of known and previously putative human isomiRs, I show that a novel subset, that includes the non-canonical miR-451, are ubiquitously modified.
In Chapter 5 I present a series of studies that have the potential to dramatically alter our understanding of miRNA biogenesis and function. Using more 30 small RNA sequencing datasets, which includes data of small RNAs associated with nuclear localized Argonaute 2 (AGO2), as well as nuclear and cytoplasmic small RNA pools, I show that miRNAs are highly abundant and associated with AGO2 in the mammalian cell nucleus, and that sdRNAs are also associated with nuclear localized AGO2. Furthermore, I show that protein-coding mRNAs are processed into small RNAs that mimic miRNAs. These miRNA-like RNAs (miLRNAs) show a pronounced 5’ end adenosine bias, are processed by DICER1 and loaded onto the miRNA effector protein, AGO2. The identification of hundreds of mRNA-derived miLRNAs in human and mouse suggests this is widespread phenomenon and hints at the existence of an additional layer of small RNA regulation embedded in genes genome-wide.
Finally, in Chapter 6 I discuss the implications of these findings and provide some perspectives on the ever-expanding small RNA field.
In summary, the small RNA world is vast and complex and is yet to be fully characterized and under- stood. This thesis further advances our insights into this complexity and provides a preliminary path to approach the discovery and characterization of novel classes of small RNAs.