Thesis Type: Postgraduate
Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey
Approval Date: 2017
Student: OĞUZHAN BEĞİK
Co-Supervisor: SREEPARNA BANERJEE, AYŞE ELİF ERSON BENSANAbstract:
Alternative polyadenylation (APA) is the selection of proximal or distal poly(A) signals on pre-mRNAs. APA has been implicated in many cellular processes, including differentiation. Resulting APA isoforms may have different stability or localization, which may eventually alter the protein function. Therefore, it is important to reveal APA isoforms to better understand post-transcriptional mechanisms in development. In this study, we aimed to investigate APA isoforms in an enterocyte differentiation model, Caco-2 cells. Enterocyte differentiation take place on the axis from colon crypt to villus to produce enterocytes from the intestinal stem cells. Caco-2 cells are derived from colon adenocarcinoma and are able to undergo spontaneous enterocyte differentiation upon confluency. Earlier, we have developed APADetect tool which uses microarray gene expression data to analyze APA events. We used APADetect in order to analyze the APA events in differentiating Caco2 cells. We identified 91 3’UTR lengthening and 43 3’UTR shortening events in differentiated Caco2 cells compared to proliferating Caco-2 cells. APA events were mostly enriched for biological processes such as enzyme binding, endocytosis and RNA processing. To begin investigating the functional significance of APA isoforms, we have looked into availability or loss of conserved miRNA binding sites on APA isoforms. Interestingly, we found an enrichment of miRNA binding sites close to the active poly(A) sites at the end of the mRNAs, which may allow easier access to miRNAs. Next, we began confirming the in silico results by real time RT-PCR (RT-qPCR) using proliferating and differentiated Caco-2 cells. Overall our approach serves as a platform for novel gene discovery in differentiation studies where conventional gene expression analysis may have overlooked 3’UTR isoforms.