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Christodoulou, N. 
ORCID: 0000-0002-7802-0904,
2023.
Regulation of microRNA activity and intestinal stem cell properties by IWS1.
PhD, Nottingham Trent University.
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Niki Christodoulou, 2023.pdf - Published version Download (25MB) | Preview |
Abstract
MicroRNAs are small single-stranded non-coding RNAs that regulate gene expression post-transcriptionally, by targeting the 3‘-untranslated region (3’-UTR) of mRNAs. The regulation of microRNAs at the transcriptional level is well-studied, however, how their activity is regulated remains largely elusive. The family of Akt serine/threonine protein kinases, comprised of Akt1, Akt2 and Akt3, regulates essential cell functions including metabolism, survival, proliferation, and migration. We have previously shown that IWS1 (Interacts with Spt6), a factor involved in mRNA splicing and nuclear export, is phosphorylated specifically by Akt1 and Akt3 at Ser720/Thr721.
Here, RNA extracts from NCI-H522 lung cancer cells, transduced with shIWS1 and reconstituted with wild type (Ser720/Thr721-IWS1) or phosphorylation-deficient IWS1 (Ala720/Ala721-IWS1) were subjected to RNA and microRNA sequencing. Bioinformatic analysis suggested that microRNAs expressed at similar levels in the two cell types have a different impact on their mRNA targets. microRNA activity reporter assays and western blot analysis for microRNA targets in these cells revealed increased microRNA activity in Ala720/Ala721-IWS1-expressing cells. Our findings were verified in a second cell line, the non-transformed immortalized colonic epithelial cells NCM460. microRNA activity reporter assays, western blot analyses and cell growth assays showed that microRNA effects are enhanced in cells expressing Ala720/Ala721-IWS1. Immunoprecipitation and proximity ligation assays showed that IWS1 interacts with proteins known to be associated with RNA-induced silencing complex (RISC), and this interaction is enhanced by Akt-mediated IWS1 phosphorylation. To evaluate the dependence of IWS1/RISC interaction on IWS1 phosphorylation, we employed Akt1-/-Akt2-/-Akt3-/- immortalized mouse lung fibroblasts, transduced with myc-Akt1 or myc-Akt2 or the empty retroviral vector. Immunoprecipitation experiments in these cells, before and after treatment with IGF1, revealed that IWS1 interacts with the RISC components specifically in Akt1- expressing cells upon Akt1 activation by IGF1. Using intestinal epithelial cells from Iws1 intestinal stem cell (ISC)-specific KO mice and human colonocytes, we found that Iws1 and IWS1 phosphorylation regulates the maintenance and differentiation of ISCs.
Overall, our data demonstrate that IWS1 interacts with RISC in a conserved Akt1- dependent manner and regulates the activity of microRNAs with significant implications in effects on cellular properties.
| Item Type: | Thesis | ||||||||||||||||
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| Creators: | Christodoulou, N. | ||||||||||||||||
| Contributors: |
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| Date: | September 2023 | ||||||||||||||||
| Rights: | The copyright in this work is held by the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the author(s) of the Intellectual Property Rights. | ||||||||||||||||
| Divisions: | Schools > School of Science and Technology | ||||||||||||||||
| Record created by: | Jeremy Silvester | ||||||||||||||||
| Date Added: | 28 Jun 2024 13:10 | ||||||||||||||||
| Last Modified: | 28 Jun 2024 13:10 | ||||||||||||||||
| URI: | https://irep.ntu.ac.uk/id/eprint/51649 |
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