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The integrity of our DNA genetic code is constantly under threat from a variety of different endogenous and exogenous sources. Failure to accurately repair damage to our DNA can lead to the development of diseases such as cancer. In order to maintain genomic stability, cells possess a network of highly regulated cellular processes which act together to repair DNA, known as the DNA damage response. Many different factors contribute to the repair of DNA lesions, including different proteins and RNA species. A central player in the repair of DNA double strand breaks, a particularly toxic type of DNA damage, is a protein known as 53BP1. The recruitment of 53BP1 to double strand breaks is a highly regulated process, and 53BP1 is constitutively bound by the inhibitor protein TIRR. When a double strand break occurs, TIRR and 53BP1 dissociate, allowing 53BP1 to engage chromatin at the break and function in repair. In Chapter 3 of this thesis, we explore the mechanism of this dissociation, identifying a role for RNA which is transcribed at double strand break itself. We also further characterise the role of TIRR in the wider DNA damage response, firstly in Chapter 4, where we explore the function of TIRR RNA binding in the regulation of RNA processing, and in Chapter 5, where we perform a proximity labelling experiment to identify novel interactors of TIRR. Taken together, this thesis characterises the function of the DNA repair factor TIRR, both as a 53BP1 negative regulator, and as an RNA-binding protein.

Vault RNA are small non-coding polymerase III transcribed RNA, which are a crucial com- ponent of a large ribonucleoprotein complex called Vault. Despite only 5% of vault RNA being associated with the Vault complex, little is known about their Vault-independent cellular roles. Here I show that vtRNA are processed by Dicer into functional small vault RNA (svtRNA) fragments that associate with Argonaute 2. Nascent RNA sequencing revealed the highest number of predicted svtRNA1-2 target genes among up-regulated genes, in AGO2 and Dicer Knock Down cells, compared to other svtRNA. svtRNA1-2 target sites were detected predominantly in the intronic regions. Genomic deletion of vtRNA1-2 results in the severe inhibition of proliferation and up-regulation of genes sig- nificantly associated with cell membrane physiology, including ERBB4 and cell adhesion. Finally, I show that svtRNA1-2 hybridises to target genes in a dual-region complemen- tarity and loop protrusion is required for intron targeting. These results show a novel small vault RNA1-2 with a role in gene expression regulation, possessing unique molecular features for targeting genes in introns leading to nascent RNA silencing.

Endogenous small non-coding RNAs (sncRNAs) are employed in mammalian cells to negatively regulate gene expression in a sequence-specific manner. This phenomenon is known as RNA interference (RNAi). Depending on the molecular mechanism, RNAi can be categorised into post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). PTGS involves direct cleavage of mature messenger RNA (mRNA) or destabilisation of mRNA and/or translational repression in the cytoplasm. TGS is mediated by the establishment of heterochromatin at target loci in the nucleus. Transfer RNAs (tRNAs), which are essential for protein synthesis, have recently been identified as a source for a novel class of regulatory sncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene regulation remain largely elusive. By using a combination of unbiased bioinformatic prediction and classic molecular biology methodologies, I have studied the role of tsRNAs in the regulation of gene expression. Here I propose that Dicer is involved in tsRNA biogenesis and Dicer-dependent tsRNAs can promote gene silencing through a mechanism that is distinct from well-known PTGS and TGS. I show that Dicer-dependent tsRNAs may lead to gene silencing by targeting nascent transcripts, a mechanism termed nascent RNA silencing (NRS). Finally, I explored the therapeutic potential of tsRNAs and demonstrated the silencing of multiple genes by employing tsRNA mimics.

Polyadenylation signals in yeast are not very well defined and are believed to be largely degenerate. Here, we present a computational and experimental genome-wide analysis of polyadenylation signals in Schizosaccharomyces pombe (S. pombe), identifying the canonical AATAAA motif as the most frequent and functional signal. RNA-Seq data from cells grown under various physiological conditions were used to map 3’UTRs, which classify as commonly heterogenic. We have shown that many genes have alternative 3’UTRs. Our results are summarised and can be accessed in a user-friendly online database Pomb(A). It has been shown that convergent genes require trans elements, like Cohesin, for efficient transcription termination. We demonstrate that convergent genes lacking Cohesin are generally associated with longer overlapping transcripts. Furthermore, we analysed ChIP-chip data of Rad21 and Mis4 as well as other Cohesin and loading complex subunits and show that regions of Rad21/Mis4 co-localisation are generally associated with highly transcribed genes. They are also cohesive, while sites with Rad21 only are less cohesive. Rad21/Mis4 co-localisation sites are in close proximity to annotated origins of replication, suggesting that cohesive sites may facilitate replication. microRNAs (miRNAs) are well studies in higher eukaryotes and participate on post-transcriptional gene silencing by degrading target mRNA or blocking translation. It is believed that miRNAs do not exist in yeast. We reanalyzed miRNA presence in yeast using recently available small RNA data sets. Potential miRNA genes and targets in S. pombe were computationally predicted based on the described alternative 3’UTR data and further experimentally tested. Dicer is an enzyme, which recognizes long dsRNA substrates and cleaves them into siRNA e↵ector molecules, essential for gene silencing. Dicer has been thought to be a purely cytoplasmic protein. However, we employed ChIP-Seq and dsRNA RNA-Seq data to show that Dicer localises in the nucleus of mammalian cells and associates with the chromatin on numerous loci. Furthermore, we present evidence that Dicer processes long dsRNA into siRNA in the nucleus and the lack of Dicer causes the accumulation of long dsRNA. This consequently induces the interferon response pathway, which ultimately leads to apoptosis and cell death.