Alternative splicing (AS) generates different mRNAs leading to production of protein isoforms with different functional properties. Since more than 90% of the human genes undergo AS, it plays a major role in the generation of human proteomic diversity. There is now ample evidence that just as AS is important for normal physiology, so altered AS is important for cancer. Altered AS in tumors is frequently due to changes in the levels of AS regulators leading to combined disruption of tumor suppressor genes and activation of oncoproteins involved in tumor establishment, progression and in resistance to therapeutic treatments. Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays a crucial role in tumor development by allowing oxygen and nutrients to reach proliferating cancer cells. In recent years, angiogenesis has been deeply investigated because its inhibition represents a promising anti-cancer therapeutic modality. However, all attempted strategies to date have shown modest therapeutic effects indicating that tumor angiogenesis is a more complex phenomenon than previously anticipated. Hence, a better understanding of the mechanisms sustaining growth of tumor vessels will be crucial to identify novel and effective anti-angiogenic therapies for cancer treatment. Traditionally, the molecular pathways involved in angiogenesis have been suggested to act primarily through regulation of transcription. Recently, the emphasis on crucial events of gene expression is changing, as many post-transcriptional programs cooperate to promote vascular development. For the first time, our group found that the formation of vascular lumen during angiogenesis is controlled at post-transcriptional level by the AS factor Nova2, previously considered neural cell-specific. Through AS of target exons affecting the Par complex and its regulators, Nova2 controls the establishment of endothelial cell polarity, a prerequisite for vascular lumen organization. Consequently, Nova2 in vivo ablation causes vascular lumen formation defects, reminiscent of aberrant morphology of the tumor vasculature. By performing RNA-seq of Nova2 depleted or overexpressing endothelial cells (ECs), I identified novel Nova2-mediated AS exons belonging to factors involved in angiogenesis or vascular development suggesting that Nova2 controls an essential layer of vascular gene expression regulation. Among the novel identified Nova2 targets in ECs there is Ptbp2, an AS factor until now considered restricted to brain and testis. Notably, I demonstrated that Nova2 functions to repress Ptbp2 expression in ECs by promoting skipping of Ptbp2 exon 10 leading to the production of a Ptbp2 transcript degraded by the NMD (Non-sense Mediated mRNA Decay) pathway. Importantly, I found that Nova2 dependent AS regulation of Ptbp2 is specifically conserved in zebrafish endothelium. Collectively, my results reveal a hierarchy of splicing factors that integrate splicing decisions during angiogenesis.
Lo splicing alternativo (AS) genera diversi mRNAs che portano alla produzione di isoforme proteiche con diverse proprietà funzionali. Poiché più del 90% dei geni umani subisce AS, esso svolge un ruolo importante nell’aumentare la capacità codificante del nostro genoma. E’ stato ampiamente dimostrato che AS è fondamentale per regolare numerosi processi cellularI; inoltre una sua deregolazione ha un ruolo chiave nella progressione tumorale. L’alterazione di AS che si osserva nei tumori è spesso causata da cambiamenti nei livelli di espressione di fattori di splicing; questo porta alla inattivazione di oncosoppressori e all’attivazione di oncoproteine coinvolte nello sviluppo e progressione della neoplasia, nonché nella resistenza ai trattamenti terapeutici. L'angiogenesi, la crescita di nuovi vasi sanguigni da vasi preesistenti, svolge un ruolo cruciale nello sviluppo del tumore, consentendo all’ossigeno e alle sostanze nutritive di raggiungere le cellule tumo-rali proliferanti. Negli ultimi anni, il processo di angiogenesi è stata molto studiato poichè la sua inibizione rappresenta una promettente strategia terapeutica contro il cancro. Tuttavia, tali terapie fino ad oggi hanno mostrato solo modesti effetti terapeutici indicando che l'angiogenesi tumorale è un fenomeno molto più complesso di quan-to precedentemente previsto. Quindi, una migliore comprensione dei meccanismi molecolari che sostengono la crescita dei vasi tumorali sarà fondamentale per individuare nuove e più efficaci terapie anti-angiogenetiche per il trattamento del cancro. Fino ad ora i meccanismi molecolari coinvolti nell’angiogenesi so-no stati principalmente investigati a livello della regolazione genica trascrizionale. Recentemente però l’importanza dei meccanismi di regolazione genica post-trascrizionale durante lo sviluppo vascolare è stata rivalutata. Per la prima volta, il nostro gruppo ha dimostrato che la formazione del sistema vascolare è controllata a livello post-trascrizionale dal fattore di AS Nova2, precedentemente considerato espresso solo dalle cellule neuronali. Attraverso la regolazione di eventi di AS di geni codificanti per membri del complesso Par e per sue proteine regolatrici, Nova2 controlla l’acquisizione della polarità da parte della cellule endoteliali (ECs) e l’organizzazione del lume vascolare durante l’angiogenesi. L’assenza di Nova2 in vivo causa difetti nella formazione del lume vascolare e questo fenotipo ricorda, sotto alcuni aspetti, la morfologia aberrante della vascolatura tumorale. Utilizzando il sequenziamento massivo dell’RNA estratto da cellule ECs deplete o overesprimenti Nova2, ho identificato nuovi esoni regolati mediante AS da Nova2 in endotelio; molti di questi esoni appartengono a geni coinvolti nell’angiogenesi o nello sviluppo vascolare, suggerendo che Nova2 ha un ruolo fondamentale nel regolare la biologia delle ECs che compongono i vasi sanguigni. Tra i nuovi bersagli di Nova2 che ho identificato c’è Ptbp2, un altro fattore di AS la cui espressione era stata fino ad ora considerata ristretta solo al cervello e al testicolo. Ho dimostrato che Nova2 reprime l’espressione di Ptbp2 nelle ECs promuovendo l’esclusione dell’esone 10 e la produzione di un trascritto di Ptbp2 che viene degradato attraverso il processo di NMD (“Non-sense Mediated mRNA Decay”). Ho trovato che la regolazione di Ptbp2 è conservata specificatamente nell’endotelio del pesce zebra. I risultati da me ottenuti rivelano una gerarchia di fattori di splicing che integrano le decisioni di splicing alternativo durante l’angiogenesi.
An RNA map of Nova2-regulated alternative splicing in endothelial cells
DI MATTEO, ANNA
2017-01-17
Abstract
Alternative splicing (AS) generates different mRNAs leading to production of protein isoforms with different functional properties. Since more than 90% of the human genes undergo AS, it plays a major role in the generation of human proteomic diversity. There is now ample evidence that just as AS is important for normal physiology, so altered AS is important for cancer. Altered AS in tumors is frequently due to changes in the levels of AS regulators leading to combined disruption of tumor suppressor genes and activation of oncoproteins involved in tumor establishment, progression and in resistance to therapeutic treatments. Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays a crucial role in tumor development by allowing oxygen and nutrients to reach proliferating cancer cells. In recent years, angiogenesis has been deeply investigated because its inhibition represents a promising anti-cancer therapeutic modality. However, all attempted strategies to date have shown modest therapeutic effects indicating that tumor angiogenesis is a more complex phenomenon than previously anticipated. Hence, a better understanding of the mechanisms sustaining growth of tumor vessels will be crucial to identify novel and effective anti-angiogenic therapies for cancer treatment. Traditionally, the molecular pathways involved in angiogenesis have been suggested to act primarily through regulation of transcription. Recently, the emphasis on crucial events of gene expression is changing, as many post-transcriptional programs cooperate to promote vascular development. For the first time, our group found that the formation of vascular lumen during angiogenesis is controlled at post-transcriptional level by the AS factor Nova2, previously considered neural cell-specific. Through AS of target exons affecting the Par complex and its regulators, Nova2 controls the establishment of endothelial cell polarity, a prerequisite for vascular lumen organization. Consequently, Nova2 in vivo ablation causes vascular lumen formation defects, reminiscent of aberrant morphology of the tumor vasculature. By performing RNA-seq of Nova2 depleted or overexpressing endothelial cells (ECs), I identified novel Nova2-mediated AS exons belonging to factors involved in angiogenesis or vascular development suggesting that Nova2 controls an essential layer of vascular gene expression regulation. Among the novel identified Nova2 targets in ECs there is Ptbp2, an AS factor until now considered restricted to brain and testis. Notably, I demonstrated that Nova2 functions to repress Ptbp2 expression in ECs by promoting skipping of Ptbp2 exon 10 leading to the production of a Ptbp2 transcript degraded by the NMD (Non-sense Mediated mRNA Decay) pathway. Importantly, I found that Nova2 dependent AS regulation of Ptbp2 is specifically conserved in zebrafish endothelium. Collectively, my results reveal a hierarchy of splicing factors that integrate splicing decisions during angiogenesis.File | Dimensione | Formato | |
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PhD Thesis Anna Di Matteo.pdf
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