Transcription Start Site selection within a single cluster and G quadruplex structures: a novel mechanism regulating gene expression

Surani, A.A. ORCID: 0000-0002-5180-9075, 2021. Transcription Start Site selection within a single cluster and G quadruplex structures: a novel mechanism regulating gene expression. PhD, Nottingham Trent University.

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The 5' untranslated region (UTR) in a messenger RNA (mRNA) can greatly influence translation. Depending on where the transcription starts, the 5’ UTR will contain (or not) regulatory elements that can modulate mRNA translation. Although studies have demonstrated the effect of differential transcription start sites (TSSs) usage on translation efficiency, these are mainly restricted to TSSs in different clusters many nucleotides apart. However, it is currently unknown if there is any relevance to the TSS variation within a single cluster. Here, we present our findings on single cluster TSS-mediated regulation of protein expression, using mainly AGAP2 as an example.

Using 5' RLM-RACE, we have identified different TSSs usage for AGAP2 mRNA in chronic myeloid leukaemia (CML) and prostate cancer (PC) cell lines, giving rise to populations of transcripts with variable lengths of 5' UTR. The population of longer 5' UTR were relatively higher in CML cell lines (P < 0.05), and those extra nucleotides contained the consensus sequence for a G-quadruplex (G4). The G4 formation was verified by CD spectroscopy. Additionally, we developed an immunoprecipitation method termed 'GRIP' [G4 RNA Immunoprecipitation] and demonstrated the existence of these RNA secondary structures in the living cells.

To study the impact of the longer 5' UTR and the G4 on translation efficiency, we cloned three 5' UTR isoforms (shorter, longer and mutated-longer version) into a bicistronic plasmid and reported a significant decrease in luciferase activity by the G4 in the longer 5' UTR (P < 0.001). This result coincides with the discrepancy noted in AGAP2 mRNA and protein levels in these cell lines. Furthermore, polysome fractionation studies also confirmed that mRNA with longer 5' UTR associated less prominently with polyribosomes (P < 0.001).

Our bioinformatics pipeline has identified 4,920 transcripts in the FANTOM database that contained putative G4 sequences between the major and upstream TSSs within the same TSS cluster. By integrating the NCI-60 microarray and SWATH-MS database, we curated a list of genes that displayed discrepancies in RNA and protein expression with a significantly higher level of G4 forming TSS; and validated our findings in another gene target (HK1). This highlights that the TSS-G4 mediated mechanism is not only limited to AGAP2 expression regulation but is also implicated in controlling the expression of other genes.

Item Type: Thesis
Creators: Surani, A.A.
Date: May 2021
Rights: This work is the intellectual property of 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 in the owner(s) of the Intellectual Property Rights.
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 07 Sep 2021 08:16
Last Modified: 04 Jul 2022 15:31

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