Insight into the function of microRNAs and other small RNAs of diverse origin

Abstract

MicroRNAs are small non-coding RNA molecules which have become appreciated over the last decade as important regulators of gene expression. Functional microRNAs (miRNAs) are bound to Argonaute, the core protein of the RNA induced silencing complex (RISC). They control gene expression by guiding RISC to target mRNAs causing RNA degradation or translational repression. Recent advances in next-generation sequencing technology have greatly increased the pace of molecular research. Nowhere is this more evident than in the field of RNA and microRNA with over 34,000 being identified (miRBase, 2014). However, a challenge of the field is to follow this rate of discovery with functional validation, which is often laborious and expensive. The underlying aim of this thesis is to better understand the functionality of miRNAs and other small RNAs of endogenous and exogenous origin. Crucial to this aim was the application of Argonaute HITSCLIP (High throughput sequencing of cross-linking immunoprecipitation) as a high throughput method of assessing the function of microRNAs, which was supported with other genomic, molecular and cellular approaches. Beginning with an introduction to the field of microRNAs (Chapter 1) and review of the experimental and genomic techniques available for this purpose (Chapter 2), Chapter 3 presents the use of Argonaute HITSCLIP to assess the functionality of microRNA mimic and inhibitors. The findings reveal that the majority of transfected RNA is not functionally bound to Argonaute but rather resides in non-functional pools within lysosomes and associated vesicles. This has important implications for the use of these common tools for miRNA overexpression or inhibition. Chapter 4 aims to interrogate theories of non-canonical miRNA biogenesis. Using RNA-seq in MDA-MB-231 breast cancer cells we observed defined small RNAs on a transcriptomewide scale including small RNAs from tRNAs, snoRNAs, vault RNAs, yRNAs, and rRNAs. Many of which are specifically Argonaute bound. To test the ability of these identified small RNAs to silence target RNAs we used reporter assays with perfectly antisense target sites cloned down stream of a luciferase reporter and antisense oligonucleotides for small RNA knockdown. From these analyses, we have concluded that the repertoire of small RNAs produced from hairpin secondary structures is not restricted to canonical miRNA precursors and many of these are specifically enriched in Argonaute immunoprecipitates. However, we emphasise that a miRNA’s functionality is relative to its abundance within RISC, and the vast majority of non-canonical miRNAs such as those produced from snoRNAs are only comparable in abundance to lowly expressed miRNAs. Although we do identify novel noncanonical miRNAs which are of moderate abundance in these cells and have the ability to repress the targets at endogenous expression. We also present that there are highly abundant small RNAs which have been postulated to act as miRNAs, such as small RNAs from y3- RNA, Chr6.tRNA166, 5s-rRNAs and U3 snoRNAs but do not repress their target despite being as abundant as miRNAs which do. In summary, this work provides insight to the function of small RNAs from diverse genomic origins as well as transfected siRNAs.