Screening Regulatory Landscape of Sialic Acid Reveals Novel microRNA Biology

• Author / Creator

  Jamechenarboo, Faezeh

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

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

  co-upregulation of a-2,3-sialyltransferases, ST3GAL1, ST3GAL2 and their glycoprotein substrate

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

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

• Subjects / Keywords

  • miRNA
  • upregulatory miRNA
  • sialic acid
  • CD98hc
  • glycosylaion
  • microRNA
  • miRNA regulatory network
  • ST6GAL1
  • sialylation

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-5y2e-ze36

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.