Alternative splicing (AS) is a process by which different protein isoforms can be generated from a single pre-mRNA transcript; these different isoforms can be characterised by either a different tissue specificity, function, structure, stability, or subcellular localisation. Given its importance in expanding protein repertoire of complex organisms, AS is tightly regulated by the combined action of cis-acting regulatory elements on the pre-mRNA and trans-acting splicing factors. Notably, AS alterations are involved in different human pathologies, including cancer development and progression. Fundamental for the tumour progression is angiogenesis, the process by which new blood vessels are formed from pre-existing ones. Angiogenesis is defined as an hallmark of cancer since it allows oxygen and nutrients to reach proliferating cancer cells and provides neoplastic cells with the metastatic route to colonize distant organs. In the past our laboratory has demonstrated that the AS regulator Nova2, previously considered as neuronal cells-specific, is also expressed in endothelial cells (ECs) of the blood vessels, where it plays fundamental roles during the development of the vascular system. In particular, we found that Nova2 controls the definition of the apical-basal polarity of ECs, a key step for the formation of vascular lumen in angiogenesis. Through RNA-Seq experiments of Nova2 gain and loss of function ECs, our laboratory also identified novel genes regulated through AS by Nova2. Among Nova2 targets in ECs we found Ptbp2, another AS factor able to bind pyrimidine-rich RNA motifs in its regulated mRNAs, that encode for factors involved in neural cell polarity and neuronal differentiation. In ECs, we found that Nova2 negatively regulates Ptbp2 levels by promoting the expression of an instable Ptbp2 mRNA degraded through the non-sense mediated mRNA decay pathway. To gain comprehensive insight in the function of Ptbp2 in angiogenesis, in collaboration with IFOM Institute of Milan, we generated stable inducible Ptbp2 overexpressing ECs. Starting from the bioinformatic analysis of our RNA-Seq data, we identified 432 novel genes regulated through AS by Ptbp2 in ECs. Remarkably, Gene Ontology analysis also showed a significant enrichment for genes involved in signalling cascades, in cell polarisation and cytoskeletal remodelling. In this thesis, I focused my attention on the validation of 12 selected AS events identified by our RNA-Seq experiment. In particular, by using RT-PCR analysis with total RNA extracted from different ECs overexpressing Ptbp2 and control ECs, I demonstrated the dependence of these AS events by Ptbp2 expression levels. Remarkably, through bioinformatic analysis, I also identified putative Ptbp2 binding sites near the regulated AS exons, suggesting that these genes could be regulated by Ptbp2 through a direct association. Collectively, my results identified novel pathways regulated through AS process, which could be important for EC biology during angiogenesis.
Lo splicing alternativo è un processo attraverso il quale diverse isoforme proteiche possono essene generate a partire da un singolo trascritto primario (pre-mRNA); queste proteine possono essere caratterizzate da una differente specificità tissutale, funzione, struttura, stabilità o localizzazione cellulare. Vista la sua importanza nell’espandere la capacità codificante dei genomi degli organismi complessi, lo splicing alternativo viene finemente regolato dall’azione combinata di elementi regolatori in cis nel pre-mRNA e da molteplici fattori di splicing. Considerata l’importanza dello splicing alternativo non è sorprendente che le sue alterazioni siano coinvolte in numerose patologie umane, fra cui lo sviluppo e la progressione del cancro. Uno dei processi fondamentali per la progressione tumorale è l’angiogenesi, meccanismo per cui nuovi vasi sanguigni vengono a formarsi a partire da vasi preesistenti. L’angiogenesi viene definita come uno degli hallmarks del cancro poiché i vasi sanguigni consentono all’ossigeno e ai nutrienti di raggiungere le cellule tumorali proliferanti, oltre che fornire loro la via per formare metastasi in altri organi. In passato il nostro laboratorio ha dimostrato che il regolatore dello splicing alternativo Nova2, per molto tempo considerato specifico dei neuroni, è anche espresso nelle cellule endoteliali (ECs) che compongono i vasi sanguigni e che Nova2 svolge un ruolo fondamentale durante lo sviluppo del sistema vascolare. In particolare, abbiamo mostrato che Nova2 controlla la definizione della polarità apico-basale delle ECs, un processo fondamentale per la formazione del lume vascolare durante l’angiogenesi. Tramite esperimenti di RNA-Seq con RNA totale estratto da ECs sovra-esprimenti o deplete di Nova2, il nostro laboratorio ha identificato nuovi geni regolati tramite splicing alternativo da Nova2. Tra questi abbiamo identificato Ptbp2, un altro fattore di splicing alternativo capace di legare sequenze ricche di pirimidine nei suoi pre-mRNA targets e che codificano per proteine coinvolte nell’acquisizione della polarità e nel differenziamento neuronale. In ECs, abbiamo dimostrato che Nova2 regola negativamente l’espressione di Ptbp2 perché promuove l’espressione di un Ptbp2 mRNA instabile e degradato attraverso il “non-sense mediated mRNA decay”. Per capire il ruolo di Ptbp2 nell’angiogenesi, abbiamo generato ECs sovra-esprimenti in modo inducibile tale fattore di splicing. Partendo dalla analisi bioinformatica dei dati di RNA-Seq, abbiamo identificato 432 nuovi geni regolati attraverso splicing alternativo regolati da Ptbp2 in ECs. L’analisi bioinformatica ha anche evidenziato un arricchimento significativo per geni coinvolti in cascate di trasduzione del segnale, nel rimodellamento della actina del citoscheletro e nella polarizzazione cellulare. In particolare, attraverso la metodica della RT-PCR con RNA totale estratto da diverse linee endoteliali over-esprimenti Ptbp2 (vs ECs di controllo), ho dimostrato che per un numero selezionato di questi geni il loro profilo di splicing alternativo era effettivamente dipendente dai livelli di espressione di Ptbp2. Ulteriori analisi bioinformatiche hanno anche identificato siti di legame putativi per Ptbp2 accanto agli esoni alternativi, suggerendo che questi geni possano essere regolati attraverso un legame diretto da Ptbp2. Collettivamente, i miei risultati hanno consentito di identificare nuovi programmi cellulari regolati a livello di splicing alternativo che potrebbero essere importanti per regolare la biologia delle cellule endoteliali durante il processo di angiogenesi.
Novel pathways regulated through alternative splicing process in endothelial cells
TERENZANI, ELISA
2020/2021
Abstract
Alternative splicing (AS) is a process by which different protein isoforms can be generated from a single pre-mRNA transcript; these different isoforms can be characterised by either a different tissue specificity, function, structure, stability, or subcellular localisation. Given its importance in expanding protein repertoire of complex organisms, AS is tightly regulated by the combined action of cis-acting regulatory elements on the pre-mRNA and trans-acting splicing factors. Notably, AS alterations are involved in different human pathologies, including cancer development and progression. Fundamental for the tumour progression is angiogenesis, the process by which new blood vessels are formed from pre-existing ones. Angiogenesis is defined as an hallmark of cancer since it allows oxygen and nutrients to reach proliferating cancer cells and provides neoplastic cells with the metastatic route to colonize distant organs. In the past our laboratory has demonstrated that the AS regulator Nova2, previously considered as neuronal cells-specific, is also expressed in endothelial cells (ECs) of the blood vessels, where it plays fundamental roles during the development of the vascular system. In particular, we found that Nova2 controls the definition of the apical-basal polarity of ECs, a key step for the formation of vascular lumen in angiogenesis. Through RNA-Seq experiments of Nova2 gain and loss of function ECs, our laboratory also identified novel genes regulated through AS by Nova2. Among Nova2 targets in ECs we found Ptbp2, another AS factor able to bind pyrimidine-rich RNA motifs in its regulated mRNAs, that encode for factors involved in neural cell polarity and neuronal differentiation. In ECs, we found that Nova2 negatively regulates Ptbp2 levels by promoting the expression of an instable Ptbp2 mRNA degraded through the non-sense mediated mRNA decay pathway. To gain comprehensive insight in the function of Ptbp2 in angiogenesis, in collaboration with IFOM Institute of Milan, we generated stable inducible Ptbp2 overexpressing ECs. Starting from the bioinformatic analysis of our RNA-Seq data, we identified 432 novel genes regulated through AS by Ptbp2 in ECs. Remarkably, Gene Ontology analysis also showed a significant enrichment for genes involved in signalling cascades, in cell polarisation and cytoskeletal remodelling. In this thesis, I focused my attention on the validation of 12 selected AS events identified by our RNA-Seq experiment. In particular, by using RT-PCR analysis with total RNA extracted from different ECs overexpressing Ptbp2 and control ECs, I demonstrated the dependence of these AS events by Ptbp2 expression levels. Remarkably, through bioinformatic analysis, I also identified putative Ptbp2 binding sites near the regulated AS exons, suggesting that these genes could be regulated by Ptbp2 through a direct association. Collectively, my results identified novel pathways regulated through AS process, which could be important for EC biology during angiogenesis.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13398