It has recently become apparent that cells encode a large number of novel non-protein-coding genes called long non-coding RNAs (lncRNAs). Whilst the biological function of many lncRNAs remains unknown, recent evidence has suggested that lncRNAs may be important regulators of cellular growth, differentiation and may play a significant role in cancer. Epidermal growth factor (EGF) – an activator of the ERK1/2 signalling cascade – is an important spatio-temporal regulator of transcription and, ultimately, of cellular growth and movement. EGF stimulation triggers a wave-like expression of immediate-early genes (IE genes), followed by delayed-early genes (DE genes) and secondary-response genes (SR genes). Over the years, considerable effort has been made to unravel the regulatory loops downstream of EGF signalling. This study investigated whether lncRNAs are sensitive to EGF signalling and whether they play a role in the transcriptional programme associated with EGF signalling. In order to identify lncRNAs regulated by EGF signalling, I sequenced nuclear RNA in the presence or absence of EGF stimulation. RNA-seq data showed that 173 lncRNAs are upregulated by EGF, of which 89 were intergenic lncRNAs (lincRNAs). The time-dependent expression profile of EGF-upregulated lincRNAs followed the well-established expression pattern of IE genes. Finally, investigation of the expression of lincRNAs in primary breast and lung cancer cells showed that EGF-upregulated lincRNAs were differentially expressed in cancer. The EGF-dependent induction profile and cancer enrichment were particularly strong for one of the transcripts – EGF-induced lncRNA 1 (EIN1) – and I selected it for further studies. Firstly, using bioinformatics and biochemical approaches, I confirmed the non-coding status of the EIN1 transcript. Secondly, I confirmed that EIN1 transcription is ERK1/2-dependent and is independent of protein synthesis. Investigation of EIN1 expression in normal tissues showed its high enrichment in the human cardiovascular system. At the cellular level, the EIN1 transcript was predominantly found in the nucleus. Functionally, the depletion of endogenous EIN1 transcripts (using the newly developed CRISPRi approach) led to changes in the EGF-dependent transcription programme. EIN1 downregulation resulted in the addition of normally EGF-independent genes into the EGF-dependent expression programme. Collectively, these results show that EGF (via the ERK1/2 pathway) can regulate transcription of lincRNAs. The EIN1 example suggests that lincRNAs may play a crucial role in the modulation of the EGF-dependent expression programme by limiting of the scope of the programme.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:703011 |
| Date | January 2016 |
| Creators | Nowicki-Osuch, Karol Piotr |
| Contributors | Sharrocks, Andrew |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/identification-and-characterisation-of-long-noncoding-rnas-expressed-downstream-of-egfinduced-signalling-programme(fd52d235-1a50-4347-bdb1-fdba4fdb912d).html |
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