Identification of microRNA targets for Isoforms of pro-apoptotic factor BIM.

• Main Author: Hong, Hui Qi.
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2013
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/52294
• Institution: Nanyang Technological University

BCL-2-interacting mediator of cell death (BIM) is a prominent regulator of the intrinsic apoptotic pathway. Aberrant BIM expression has been implicated in a multitude of diseases. Intriguingly, post-transcriptional regulation, modulated primarily by cis-elements in the 3’untranslated regions (UTRs), accounts for approximately 60-80% of overall regulatory effects on expression of protein-coding genes. Identification of such cis-elements as microRNA (miRNA) targets in BIM 3’UTRs is therefore of intense interest, as these cis-elements are potential therapeutic targets for amelioration of aberrant BIM expression. Herein, we individually cloned the 3’UTR of BIM Exons 3 and 5, the only two possible 3’-terminal exons of all BIM-encoding transcripts, into a reporter Firefly luciferase expression plasmid. Subsequently, we introduced serial deletions in these 3’UTRs using polymerase chain reaction (PCR) mutagenesis. Comparing reporter gene expression level of constructs containing any of the wild-type BIM 3’UTRs with their mutant counterparts afforded identification of functional cis-regulatory clusters. In this preliminary study, we identified multiple cis-regulatory regions that might harbour miRNA targets; however, future studies are necessary to define the exact miRNA targets of BIM. Moreover, our study revealed that negative cis-elements within Exon 5 are enriched in the distal tandem 3’UTR, which may have important therapeutic implications.