Approches de fractionnement biochimique couplé à la transcriptomique dans l’étude systématique de la localisation subcellulaire et extracellulaire des ARNs

Several RNA transcripts acquire spatially-resolved patterns in diverse prokaryotic and eukaryotic cells, a phenomenon termed “RNA localization”. In highly polarized cells, such as neurons or certain embryos, mRNA localization provides an elegant mechanism to restrict protein expression and activity to a narrow spatiotemporal context. Diverse imaging approaches have been developed to study RNA localization, including RNA in situ hybridization and the MS2 system. While these techniques enable the visualization of RNA spatial distributions at a high resolution, they hardly allow for systematic, transcriptome-wide analyses of spatial asymmetry.   The first part of this thesis encompasses the development of biochemical, cell fractionation and extracellular milieu processing methods coupled to deep sequencing as a novel approach to study transcriptome-wide RNA localization.  These methods, collectively termed CeFra-seq (“Cell Fractionation – RNA-seq”), are propose as a complementary tool with imaging approaches.  Chapter 5 consists of a detailed technical description of the CeFra-seq methodology, along with a validation workflow and a relevant data transformation toolkit. The subsequent chapters discuss applications of these methods to investigate four outstanding questions in different biological systems.   Chapter 6 relies on CeFra-seq to explore the subcellular properties of RNAs targeted to extracellular vesicles (EVs). EVs form a group of heterogeneous nanoscopic structures delimited by a phospholipid bilayer that contain specific repertoires of protein and nucleic acids. Ubiquitous in biological fluids, EVs have been associated to intercellular communication in diverse biological contexts, notably antigen presentation and tumor progression. Yet, the mechanisms that account for the enrichment of specific RNAs in EVs remain unclear. By systematically contrasting RNA populations found in EVs with subcellular distributions, my work has revealed diverse properties linked to EV targeting, including cytosolic accessibility, RNA length and cis-acting elements.   Chapter 7 consists of an extensive comparison of the morphological and transcriptomic properties of EVs derived from several human and Drosophila cell lines. This work reveals that Drosophila EVs are smaller than their human counterparts and that they are both enriched in short, Polymerase III transcripts. Together, these results emphasize the high conservation of RNA export processes.   Chapter 8 extends subcellular fractionation approaches coupled to deep sequencing in an additional biological system: Drosophila embryogenesis. Here, the method leads to repertoires of transcripts displaying high spatial and temporal asymmetry during development. The analysis of SLBP mutants, a factor involved in histone mRNA processing, and Chk1 mutnts, a regulator of the DNA damage response, shows that the depletion of these proteins selectively hampers the expression of zygotic transcripts, identified through CeFra-seq.   Chapter 9 recounts a study of antisense transcripts derived from the histone gene locus during Drosophila embryogenesis. The expression of these non-polyadenylated RNAs fluctuates during development and depends on the protein SLBP. Here, CeFra-seq reveals that these antisense RNAs, which are strictly zygotic, co-segregate with their complementary mRNAs, hinting at the formation of double-stranded RNAs, precursors of small interfering RNAs. To follow-up on this hypothesis, I show that small RNAs derived from the histone gene locus bind to the catalytic factor of the RNA-induced silencing complex, Argonaute-2. In addition, depleting Argonaute-2 leads to a derepression of histone mRNAs. Together, these results suggest a model wherein precocious zygotic antisense transcription leads to the formation of small interfering RNAs, which contribute to the clearance of maternally deposited histone mRNAs.   Hence, this thesis reflects the development and application of a versatile approach to study RNA localization, termed CeFra-seq and described in chapter 5. The use of this method leads to functional studies aiming to investigate the properties of EV-targeted RNAs (Chapter 6) and the extent of evolutionary conservation of this targeting process (Chapter 7). The rest of the thesis exploits the CeFra-seq approach to explore the transcriptomic phenotype of SLBP depletion in Drosophila embryos (Chapter 8), as well as the properties and functions of antisense RNAs produced by the histone gene locus in early embryos (Chapter 9).

Keyphrases: exosomes, RNA localization, Posttranscriptional regulation, Systems biology, Transcriptomics, Non-coding RNAs, Extracellular vesicles, Embryonic development, Drosophila