From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C
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 associ...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2024
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| Online Access: | https://eprints.nottingham.ac.uk/78422/ |
| _version_ | 1848801079785947136 |
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| author | Dawson, Owen |
| author_facet | Dawson, Owen |
| author_sort | Dawson, Owen |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T21:01:45Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-78422 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:01:45Z |
| publishDate | 2024 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-784222024-08-13T14:24:04Z https://eprints.nottingham.ac.uk/78422/ From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C Dawson, Owen 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. 2024-07-24 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/78422/1/Owen%20Dawson_Amended%20Thesis_June%202024.pdf 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. microRNAs mirna miR-155 miR-155-5p miR-155-3p Tenascin-C Inflammation Macrophage Yes-associated protein arm-switching. |
| spellingShingle | microRNAs mirna miR-155 miR-155-5p miR-155-3p Tenascin-C Inflammation Macrophage Yes-associated protein arm-switching. Dawson, Owen From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title | From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title_full | From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title_fullStr | From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title_full_unstemmed | From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title_short | From the extracellular matrix to the microprocessor: Investigating the post-transcriptional regulation of miRNA-155 by tenascin-C |
| title_sort | from the extracellular matrix to the microprocessor: investigating the post-transcriptional regulation of mirna-155 by tenascin-c |
| topic | microRNAs mirna miR-155 miR-155-5p miR-155-3p Tenascin-C Inflammation Macrophage Yes-associated protein arm-switching. |
| url | https://eprints.nottingham.ac.uk/78422/ |