The AID/APOBEC protein family comprises a group of cytosine deaminases found in vertebrates that are capable of modifying cytosine to uracil in the context of RNA or singlestranded DNA. They exert diverse valuable physiological functions including antibody diversification and restriction of viral infection. However, off-target mutations have also been shown to contribute to cancer development, making it crucial to better understand the interactions and mechanisms that regulate AID/APOBEC activity and editing site fidelity. In this regard, a new focus on RNA as a putative regulator of AID/APOBECs has recently emerged. Regardless of whether it is used or not as a substrate for deamination, most members of the family have been shown to retain the ability to bind RNA, emphasizing a potential regulatory role for this interaction. However, little is known about AID/APOBECs RNA binding specificity. A promiscuous binding has been suggested in some cases while in vitro evidence for other members of the family indicate a certain level of specificity. Therefore, to thoroughly unravel the AID/APOBECs RNA binding specificity, in my doctoral research I applied cross-linking and immunoprecipitation (iCLIP), an unbiased technique that allows identification of protein-bound RNAs with nucleotide resolution in living cells. As a first approach, I adapted the technique for its use in yeast and probed the RNA binding of AID and APOBEC3G, revealing different degrees of preference for small structured RNAs and recognition of particular sites within them. I then expanded the analysis to mammalian cells (HEK293T) and evaluated an extended set of APOBECs finding that, even in the presence of a broader and more complex pool of RNAs, small RNAs were still significantly bound by some members of the family. Furthermore, the comparative analysis of AID, APOBEC1, APOBEC3G, APOBEC3A and APOBEC3B iCLIP data obtained in my research, revealed shared and individual preferences for certain RNAs, suggesting a degree of binding specificity among APOBECs. In summary, my thesis outlines for the first time a comprehensive analysis of the RNA binding specificity of different AID/APOBECs in vivo, including the description of novel interactions with nucleotide resolution. The results obtained are of great value and open the field for further investigation of the specific meaning and validation of each preferential binding, providing new insights into understanding the role of AID/APOBEC interaction with RNA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:767767 |
| Date | January 2019 |
| Creators | Valeiras, Brenda |
| Contributors | Rada, Cristina |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/288562 |
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