My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). All RNAs that are transcribed in the cell are initially produced as precursor RNAs, which have to undergo various steps of processing to obtain their mature form. In the cell, the genetic information encoded in the DNA is transcribed to RNA. 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. 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. 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. 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. 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. 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. 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. However, little is known about AID/APOBECs RNA binding specificity. 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. In this regard, a new focus on RNA as a putative regulator of AID/APOBECs has recently emerged. 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.

They exert diverse valuable physiological functions including antibody diversification and restriction of viral infection. 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.

Iclip technique