Screening Regulatory Landscape of Sialic Acid Reveals Novel microRNA Biology
Creators: Jamechenarboo, Faezeh
Publisher: name: University of Alberta Library
Year: 2024

<p>Sialylation is a crucial post-translational modification that covalently attaches sialic acid<br>
to glycoproteins and glycolipids, and is driven by a superfamily of enzymes called<br>
sialyltransferases (STs). Alterations in sialylation have been identified in different biological<br>
contexts including cancer biology (transformation, progression and malignancies); immunology<br>
(cellular communication, cell signaling); infectious dieases (host-pathogen interactions); and<br>
neurodegenerative disorders. The expression levels of STs directly impact the cellular sialylation<br>
content through which they in turn affect cell normal physiological and pathological status. Protein<br>
homeostasis, the balance of proteins, is tuned post-transcriptionally. microRNA (miRNA) mediate<br>
protein expression via direct interactions with corresponding transcript, mainly with 3&#39; untranslated<br>
region (3&#39;UTR). In my Ph.D. journey in the Mahal laboratory, I aim to address the query of “how<br>
human sialylation is regulated by miRNA?”<br>
In Chapter 2, the miRNA regulatory landscape of a-2,6-sialyltransferases (i.e., ST6GAL1<br>
and ST6GAL2) were profiled using a ratiometric fluorescence assay called “miRFluR”.<br>
Unexpectedly, the analysis reveals a bidirectional tuning of protein expression by miRNA: the<br>
inhibition of translation and protein upregulation. The observed miRNA-mediated protein<br>
expressions were validated across different cancer cell lines for their impact on protein, mRNA of<br>
ST6GAL1 and ST6GAL2, and a-2,6-sialylation levels. Direct miRNA: 3&#39;UTR interactions,<br>
predicted by RNAhybrid or Targetscan, confirmed via mutating the interacting nucleotides and<br>
testing WT and mutant pFmiR sensors using miRFluR assay. The scope of the newly discovered</p>

<p>miRNA function, protein upregulation in actively dividing cells, were then expanded to co-<br>
upregulation of functionally associated proteins by microRNA, a story described in Chapter 3 for</p>

<p>co-upregulation of a-2,3-sialyltransferases, ST3GAL1, ST3GAL2 and their glycoprotein substrate</p>

<p>CD98hc. The three proteins were recently found to be functionally correlated in melanoma. In<br>
Chapter 3, my analysis showed that microRNA cooperatively upregulate either<br>
CD98hc:ST3GAL1 or CD98hc:ST3GAL2 protein pairs in melanoma cell lines. The direct impacts<br>
by miRNA on the protein co-regulation is confirmed by mutational analysis of the corresponding<br>
3&#39;UTRs.</p>

<p>In Chapter 4, the miRNA modulatory axis of another member of a-2,3-sialyltransferases,<br>
ST3GAL5, was examined. While its miRNA regulation has been partially investigated by the<br>
Mahal laboratory, I screened the potential human miRNA interactome over ST3GAL5 3&#39;UTR via<br>
the high-throughput miRFluR assay. ST3GAL5 initiates the ganglioside biosynthetic pathway by<br>
providing GM3 substrate for other glycosyltransferases to extend the glycan chain on lipids.<br>
Intriguingly, miRNA were identified to mediate both ST3GAL5 expression and cell surface GM3<br>
epitope in different cancer cell lines.<br>
Apart from sialyltransferases, a key enzyme that impacts cellular sialylation levels is<br>
CMAS. This enzyme is responsible for providing activated substrate (CMP-sialic acid) for<br>
sialyltransferases, accordingly, it can impact both cellular CMP-sialic acid content and sialylation.<br>
In Chapter 5, miRNA were identified to both down- and up-regulate CMAS expression as well as<br>
sialylation via direct miRNA: CMAS 3&#39;UTR interaction.<br>
Together, my Ph.D. dissertation may help to expand our current understanding of the<br>
regulation of sialylation as well as microRNA biology.</p>