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High Throughput Analysis of microRNA Regulation of MGAT3
- Author / Creator
- Zohora, Fatema Tuz
Studies related to miRNAs and their ability to alter protein expression have gained much attention in recent years. miRNAs are short single stranded non-coding RNAs (~20 nts) that function in RNA silencing and post-transcriptional modification of gene expression. These short non-coding RNAs primarily bind to the 3’-untranslated region (UTR) of target mRNA transcript. This interaction can cause either translational repression or inhibition mainly via mRNA degradation. miRNAs can also bind to the 5’-untranslated region of target mRNA and regulate protein expression in humans, though the general role of 5’-untranslated region in miRNA mediated regulation still remains unknown.
In the field of glycobiology, miRNAs are now given special consideration as they now have been proven to be a key regulator in many glycosylation pathways. N-linked branching of glycans is one of the important glycosylation pathways in eukaryotes and is critical for both the structural and functional integrity of many eukaryotic proteins. Several glycosyltransferase enzymes mainly encoded by MGAT genes are involved in the synthetic pathway of N-linked branching. MGAT3 (β-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase) enzyme is responsible for the addition of a bisecting GlcNAc to the core mannose residue of complex or hybrid N-glycans. The addition of bisecting GlcNAc is a unique modification of complex N-glycans that has been reported to play important role in signal transduction and growth factor signaling pathway, tumor progression and metastasis.
In this thesis, we aim to generate a comprehensive map of the regulation of MGAT3 by miRNA using a newly developed high throughput assay: miRFluR. This assay uses a genetically encoded dual-color fluorescence reporter to identify regulatory miRNA-mRNA interactions. At first we cloned 3’UTR sequence of MGAT3 into empty pFmiR backbone downstream to cerulean protein. Then we co-transfected this 3’UTR-plasmis sensor of MGAT3 with a library of 2601 miRNAs that represent the known human miRNAome in HEK293T cells. After 48 hours of transfection, we performed a ratiomatric analysis of cerulean and mCherry protein expression and established a complete map of miRNAs regulating MGAT3 protein expression.
miRFluR assay data revealed a significant number of miRNAs upregulating MGAT3 expression as opposed to their common repression mechanism. These up-regulatory miRNAs were later validated in three different mammalian cancer cell lines by Western blot, RT-qPCR, PHA-E lectin staining and site specific mutation of 3’UTR-sequence of MGAT3. Total 125 up-regulatory miRNAs were identified from the assay compare to only 15 down-regulatory miRNAs. This indicates the dominance of miRNA-mediated upregulation over MGAT3. In addition to that the up-regulatory miRs are highly enriched in cancer pathways predicting the potential role of MGAT3 in cancer. miRNA regulation of genes has been shown to predict functional roles in disease pathways. Thus mapping MGAT3 has enabled us to look deep into the functions of MGAT3 and bisecting GlcNAc in diseases.
- Graduation date
- Fall 2023
- Type of Item
- Master of Science
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