Dawson, Owen
(2024)
From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C.
PhD thesis, University of Nottingham.
Abstract
A highly controlled type of short RNA sequences called microRNAs (miRNAs) regulates cellular processes via translational repression or degradation of target mRNAs. In particular, miRNA-155 plays a central role in the macrophage inflammatory response to infection, with dysregulation of miR-155 associated with pathological inflammation.
Previous work in the Piccinini lab has identified a post-transcriptional mechanism of miR-155 regulation, showing that the extracellular matrix (ECM) glycoprotein tenascin-C (TN-C) regulates processing of the miR-155 transcript (pri-miR155) in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs). However, how this regulation occurs is yet to be elucidated.
This project aimed to identify by what mechanism TN-C regulates miR-155 processing in activated macrophages, involving examination of candidate TNC receptors, intracellular signalling pathways associated with both TN-C and miR-155 biogenesis machinery, as well as identification of pri-miRNA features associated with TN-Cs regulatory activity.
Utilising gain and loss-of-function approaches, examination of the candidate yes-associated protein (YAP) pathway, an increasingly relevant pathway in the study of cancer and inflammation, showed no association between this pathway and the post-transcriptional regulation of miR-155 by TN-C. Remarkably, however, for the first time ectopic expression of YAP was found to negatively regulate macrophage miR-155 transcription, this being linked to inhibition of the critical pro-inflammatory transcription factor NF-κB.
Additionally, knockdown of TN-C expression in steady-state RAW 246.7 macrophages found TN-C to regulate p38α and MK2, a master regulator of RNA-binding proteins, total protein abundance, but not their phosphorylation, this being attributed to an increase in p38α mRNA levels as a result of TN-C knockdown, occurring via an unknown mechanism.
Furthermore, siRNA-based knockdown analysis of candidate TN-C receptors pointed to integrin αV as potential transducer of TN-C signalling the regulation of miR-155 expression.
Finally, interrogation of RNA-SEQ data from LPS treated TNC knockout and wild type BMDMs found no significant association between specific pri-miRNAs cis-regulatory elements, known to facilitate miRNA processing, and miRNAs modulated by TN-C in a similar manner as miR-155. Interestingly, TNC knockout was found to associate with a significant decline in 5p strand mature miRNA, with a concurrent induction in the levels of the 3p strand, potential evidence of TN-C orchestrating a miRNA arm-switching event.
Overall, whilst this project rules out YAP and MK2 as molecular players in the TN-C-miR-155 pathway, it unveils two new research directions: YAP as potential regulator of miR-155 transcription independent of TN-C and, excitingly, TN-C as a potential regulator of cell-wide miRNA strand selection.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Piccinini, Anna M. |
| Keywords: |
microRNAs, mirna, miR-155, miR-155-5p, miR-155-3p, Tenascin-C, Inflammation, Macrophage, Yes-associated protein, arm-switching. |
| Subjects: |
Q Science > QP Physiology > QP501 Animal biochemistry
Q Science > QR Microbiology > QR180 Immunology |
| Faculties/Schools: |
UK Campuses > Faculty of Science > School of Pharmacy |
| Item ID: |
78422 |
| Depositing User: |
Dawson, Owen
|
| Date Deposited: |
13 Aug 2024 14:24 |
| Last Modified: |
13 Aug 2024 14:24 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/78422 |
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