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Terminal uridyl transferases: TUT4/7-mediated RNA metabolism in cancer 10.17863/CAM.63455 Medhi, Ragini 0000000227800708 Miska, Eric 000000024450576X Nascent RNA is subjected to a wide range of RNA metabolic processes such as non-templated additions of uridines at the 3′ end after it has been transcribed. These additions are catalysed by the terminal uridyl transferases TUT4 and TUT7 (TUT4/7). Defects in TUT4/7-mediated functions result in sterility, failed embryogenesis and susceptibility to viral pathogens. Additionally, TUT4/7 have been shown to be key regulators of the tumorigenic LIN28A/let-7 pathway. However, a full understanding of TUT4/7-mediated mechanisms of RNA control that impinge on tumorigenesis is still missing. In this thesis, I establish catalytic knockouts of TUT4/7 in two distinct cancer cell types to understand the mechanistic aspects of TUT4/7-mediated regulation in tumorigenesis. I observe cell type specific defects in cancer properties in the TUT4/7 double mutants. As the cell type specific differences in defects could be due to the presence or absence of LIN28A, I integrated the LIN28A cDNA in the LIN28A-negative cancer cell line. This allowed the comparison of TUT4/7-dependent gene expression changes in a LIN28A context. My findings suggest that miRNAs and mRNAs do not generally depend on LIN28A-mediated TUT4/7 regulation. Instead, I find that TUT4/7 can shape the transcriptomic landscape according to the cancer cell type independently of LIN28A. Furthermore, I provide new examples of emerging compensatory mechanisms that arise upon TUT4/7 loss. I observe that loss of uridylation results in a simultaneous gain in 3′ adenylation. The extent of gain in adenylation is miRNA-specific with some miRNAs overexpressing adenylated isomiRs upon TUT4/7 loss. This might contribute to the observed proliferative defects in the TUT4/7 double mutants. Finally, I show that TUT4 and TUT7 have non-redundant functions. I identify miRNA targets of TUT7 that are not uridylated by TUT4 and vice versa. I also present ongoing work on the development of an effective technique to identify direct targets of TUT4/7 and to gain a comprehensive view of RNA control mechanisms based on sequence specific features. Having examined the TUT4/7-mediated regulatory networks at a transcriptome level, my findings show that TUT4/7 is a promising cancer target for an ovarian cancer subtype. However, exploring the biological effects of the novel compensatory mechanisms that emerge upon TUT4/7 loss warrant further study so as to prevent deleterious consequences when targeting TUT4/7 as a potential cancer therapy. University of Cambridge 2020-09-01 Thesis Doctoral Doctor of Philosophy (PhD) eng Storm Therapeutics Ltd. https://www.repository.cam.ac.uk/handle/1810/316345 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a94e2999-cb2c-445b-a897-35a25fd6f53e/download 4616c14a1edb525f3922f5cf90ccce6f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/d0cddbfe-7946-4a3c-9b17-2b1dc085cb4e/download 353adac0d1ebdfd65ab16480263c3c87 https://www.rioxx.net/licenses/all-rights-reserved/ Cancer Tumorigenesis RNA RNA metabolism