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.

In the cell, the genetic information encoded in the DNA is transcribed to RNA. 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. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general. By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm. I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences. Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.

In the cell, the genetic information encoded in the DNA is transcribed to RNA. 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. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general. By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm. I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences. Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.

This thesis characterizes RNA footprints of several RNA-binding proteins (RBPs) thatare involved in U-insertion/deletion, A-to-I, and C-to-U RNA editing in Trypanosoma brucei. Relying on iCLIP data and biochemical methods it shows that two paralogs proteins from the MRB1 complex regulate distinct editing fates of the mitochondrial transcripts. Further, this thesis provides evidence where the combinatorial interplay of RBPs might fine-tune the levels of edited mRNA. Finally, the presented thesis adds to the growing evidence of the importance of RBPs in post-transcriptional gene regulation.

No description available.

Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.