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    Identification and characterisation of long non-coding RNAs expressed downstream of EGF-induced signalling programme.

    Nowicki-Osuch, Karol Piotr

    [Thesis]. Manchester, UK: The University of Manchester; 2016.

    Access to files

    Abstract

    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.

    Additional content not available electronically

    All of the following files are available on the CD/DVDSupplementary file 1. The summary of the differential expression analysis of the high-depth nuclear RNA-seq.Supplementary file 2. The Ingenuity Pathway analysis output for the upstream regulators of EGF-regulated genes. Supplementary file 3. The average distribution of sequencing reads around TSSs of differentially expressed genes. Supplementary file 4. The expression data for the breast cancer samples (Horvath et al., 2013).Supplementary file 5. The expression data for the lung cancer samples (Seo et al., 2012).Supplementary file 6. The summary of the differential expression analysis of the total cell RNA-seq after EIN1 depletion.Supplementary files 7 and 8. Line plot of expression of genes regulated by EGF stimulation.Supplementary file 9. Pairwise scatterplots of RNA-seq data for protein-coding genes.Supplementary files 10-12 Ingenuity Pathways Analysis summary for analysis of EGF induced data.Supplementary files 13-16 Ingenuity Pathways Analysis summary for analysis of EIN1-regulated genes.Supplementary Materials file 1. List of primers used.

    Bibliographic metadata

    Type of resource:
    Content type:
    Administered thesis
    Form of thesis:
    Traditional
    Type of submission:
    Doctoral level ETD - final
    Thesis title:
    Identification and characterisation of long non-coding RNAs expressed downstream of EGF-induced signalling programme.
    Degree type:
    Doctor of Philosophy
    Degree programme:
    PhD Wellcome Trust - Molecular and Cell Biology
    Publication date:
    2016-01-28T16:10:52
    Institution:
    The University of Manchester
    Location:
    Manchester, UK
    Total pages:
    195
    Abstract:
    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.
    Additional digital content not deposited electronically:
    All of the following files are available on the CD/DVDSupplementary file 1. The summary of the differential expression analysis of the high-depth nuclear RNA-seq.Supplementary file 2. The Ingenuity Pathway analysis output for the upstream regulators of EGF-regulated genes. Supplementary file 3. The average distribution of sequencing reads around TSSs of differentially expressed genes. Supplementary file 4. The expression data for the breast cancer samples (Horvath et al., 2013).Supplementary file 5. The expression data for the lung cancer samples (Seo et al., 2012).Supplementary file 6. The summary of the differential expression analysis of the total cell RNA-seq after EIN1 depletion.Supplementary files 7 and 8. Line plot of expression of genes regulated by EGF stimulation.Supplementary file 9. Pairwise scatterplots of RNA-seq data for protein-coding genes.Supplementary files 10-12 Ingenuity Pathways Analysis summary for analysis of EGF induced data.Supplementary files 13-16 Ingenuity Pathways Analysis summary for analysis of EIN1-regulated genes.Supplementary Materials file 1. List of primers used.
    Keyword(s):
    Thesis main supervisor(s):
    Thesis co-supervisor(s):
    Degree grantor:
    Language:
    en

    Institutional metadata

    University researcher(s):
    Academic department(s):

    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:296204
    Created by:
    Nowicki-Osuch, Karol
    Created:
    28th January, 2016, 16:10:52
    Last modified by:
    Nowicki-Osuch, Karol
    Last modified:
    29th November, 2017, 10:09:58

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