Alternative polyadenylation of Rho GTPases : a gene/cell specific process

• Autor(a) principal: Braz, Sandra Catarina Oliveira
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/14865
• topic: Biologia molecular; Diferenciação celular; Ácido ribonucleico; Ácido ribonucleico; Expressão genética; Transdução de sinal celular

Alternative polyadenylation (APA) is an important mechanism of gene regulation that occurs in 70% of eukaryotic organisms. This process comprises the formation of alternative 3’ ends of an mRNA by cleavage of the pre-mRNA and polyadenylation at different sites according to the polyadenylation signals (pAs). The choice of pAs in APA is a co-transcriptional mechanism that depends on auxiliary cis- and trans-acting factors. The usage of the proximal or the distal pAs has been related to global physiologic events. It is consensually assumed that in proliferative conditions there is preferential usage of proximal pAs, while during development and in differentiated cellular states occurs lengthening of the 3’UTRs by selection of the distal pAs. This pattern is also confirmed in brain tissues, where most of the cells are differentiated, and where it was observed a lengthening of the 3’ UTRs. However, there is not a complete switch for the distal pA, since the shortest mRNA is still expressed. Rho GTPases are key molecular switchers essential for several cellular processes, including differentiation, however nothing is known about transcriptional regulation in these genes. Therefore, we started to explore if Rho GTPases genes undergo APA. We found by 3’RACE analyses, that classical Rho GTPAses express two alternative mRNA isoforms. However during oligodendrocytes differentiation, they preferentially express the shortest mRNA isoform, and we did not observe a switch towards the distal pA usage, in contrast with the published genome-wide data obtained from brain tissues. Since Rho GTPases are tightly regulated at the protein level by GEFs and GAPs, they may not require this mode of co-transcriptional regulation. The atypical RhoBTB2, which is constitutively active, present a global induction of distal pA sites, distinct from the classical Rho GTPases. Interestingly, this pattern suggests that APA is a gene specific mechanism. As longer 3'UTRs contain more binding sites for miRNAs and RNA binding proteins (RBPs) this suggests that atypical Rho GTPases require a fine-tune regulation at the co-transcriptional level, by APA. Additionally, we showed that APA is also cell-specific, by analyzing the expression of the different mRNA isoforms of Rho GTPases in other glial cells (microglia, astrocytes) and different types of neurons (cortical, striatal and hippocampal). We observed the same APA profile for the selected Rho GTPases in all glial cells types. However, in cortical and striatal neurons we observed a lengthening in the 3’UTR Rac1 mRNA during axonal growth, which results in the increase of the total protein levels. Taken together, our results indicate for the first time that APA is a gene- and cell- specific mechanism. In addition, we have found a differential expression of both Cdc42 isoforms during OL and sciatic nerve differentiation. During in vitro OL and in vivo sciatic nerve differentiation we observed an increase in the expression ratio between Cdc42 Iso1/Cdc42 Iso2. Further, constitutive expression of Cdc42 Iso2 in OLs induces a delay in differentiation, whereas constitutive expression of Cdc42 Iso1 induces an increase in OL branching, suggesting an exacerbation of the differentiated phenotype. Thus, these observations suggest a distinct role for the different Cdc42 isoforms during OL differentiation. Overall, this thesis opens new avenues to explore in the future that can impact our understanding on the regulation of the myelination/remyelination processes.