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Origins of non-coding Y RNA family and its role in DNA replication https://doi.org/10.17863/CAM.110562 Pavlickova, Milena Krude, Torsten Y RNAs are a family of non-coding RNAs that are required for chromosomal DNA replication initiation in vertebrates. Specifically, Y RNAs play an essential role in the establishment of new DNA replication forks during G1 to S phase transition. However, the mechanism by which Y RNAs contribute to initiation of DNA replication, as well as the evolution of Y RNAs through vertebrates and invertebrates remain unknown. Y RNA upper stem domain is a minimum essential domain that conveys the Y RNA replication initiation activity. Here, I have established a single nucleotide contribution to the DNA replication activity of the upper stem domain. I have showed that the upper stem length, specific bases of its 5’-GUG-CAC-3’ motif and orientation of its G:C clamp motifs must be rigidly conserved for the DNA replication initiation activity to be preserved. Furthermore, I showed that replication factors such as ORC, Cdc6 and Cdt1 are unlikely to interact with the Y RNA upper stem domain, contrary to previous findings. Functional homologs of Y RNAs called sbRNAs, have been identified in the nematode genus *Caenorhabditis*. However, no additional Y RNAs were found in direct vertebrate ancestors, nor in any other invertebrate species. I analysed two distant invertebrate genera: the insect *Drosophila* group and a direct vertebrate ancestor, a chordate subphylum, lancelets. I demonstrated that previously established *Drosophila* Y RNA candidates do not initiate DNA replication despite possessing the upper stem domain and are in fact not part of the Y RNA family. Additionally, I successfully identified novel group of invertebrate Y RNAs in lancelets which demonstrated DNA replication initiation activity as well as high levels of expression in endogenous organisms. Therefore, in this thesis, I have further clarified the role of Y RNAs during DNA replication as well as identified ancestral Y RNAs showing that they are more prevalent during pre-vertebrate evolution than previously thought. University of Cambridge 2021-12-28 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/371353 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2945bde0-bb08-4f78-b21d-7d90386474a1/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a3b047c4-4a7f-4cc0-9155-5d200072c671/download 9f75f6bf08d368346fb4af8625401ae8 https://www.rioxx.net/licenses/all-rights-reserved/ DNA replication DNA replication origin lancelet non-coding RNAs sbRNA vertebrate DNA replication Y RNA