Regulation of MCL1 alternative polyadenylationderived mRNA isoforms by microRNAs in human T cells

• Autor(a) principal: Sousa, Ana Cláudia Faria Curinha de
• 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/10773/15280
• topic: Biologia molecular; Expressão genética; Transcrição genética; Proteínas; Ácido ribonucleico; Metabolismo celular

Polyadenylation is a fundamental processing step of mRNA maturation, essential for its export, stability and translation. Bioinformatic analyses have shown that 70% of human genes have several polyadenylation signals (pA signals) in the 3’untranslated region (3’UTR) that are used to produce multiple mRNA isoforms by alternative polyadenylation (APA). This process has a fundamental role in gene expression in a variety of cellular programs as well as in non-pathological conditions and diseases. When it occurs in the 3’UTR, APA gives rise to transcripts with different 3’UTR lengths. MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) often bind to sequences present in that region and thus mRNA isoforms with longer 3’UTRs have more sites where these regulators can bind, being more prone to regulation. miRNAs are 23 nucleotides in length post-transcriptional regulators of gene expression that have been implicated in a variety of cell conditions. In the immune system, it has been shown that upon T cell activation there is a global switch in pA signal selection from a distal to a proximal pA signal, originating mRNAs with shorter 3’UTRs and, consequently, with less miRNAs and RBPs target sites. The MCL1 (Myeloid Cell Leukemia Sequence 1) gene encodes an anti-apoptotic protein (Mcl-1), which is a member of the Bcl-2 (B cell lymphoma-2) family of apoptosis regulators. MCL1 is essential for development and maintenance of both B and T lymphocytes in animals. It has been demonstrated that MCL1 is highly regulated at both transcriptional and post-transcriptional levels. The aims of our study were to characterize the pattern of MCL1 APA and to unravel the role of miRNAs in the regulation of MCL1 APA-derived isoforms. We discovered that MCL1 produces four mRNA isoforms by the usage of four canonical pA signals located in the 3’UTR and that these pA signals are highly conserved in mammals. We verified that the longest mRNA isoform is regulated at a post-transcriptional level in activated PBMCs. We also demonstrated that the shortest 3’UTRs give rise to higher amounts of luciferase activity than the longest mRNA. Additionally, we showed that Mcl-1 protein levels increase upon PBMCs activation. This suggests that during PBMCs activation the increase in Mcl-1 protein is due to the translation of the shortest isoforms. In the second part of this study we identified miRNA-17 as a putative regulator of the longest isoform upon T cell activation. The expression of this miRNA increased upon activation of T cells and when we mutated its putative-binding site on MCL1 3’UTR we observed an increase in luciferase activity in HeLa cells. We also overexpressed miRNA-17, miRNA-29b and miRNA-92a and showed that this causes a decrease in endogenous Mcl-1 expression. From this study we conclude that the MCL1 longest APA-derived isoform is down-regulated at a post-transcriptional level upon T cell activation in order to increase Mcl-1 expression and that miRNA-17 may be the key regulator in this mechanism.