Regulation of alternative splicing by G-quadruplexes: molecular mechanisms and tools to manipulate gene expression

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G-quadruplexes (G4) play crucial roles in gene expression but their function in RNA processing and alternative splicing in particular remain poorly understood and controversial. We have shown that the pre-mRNA of the Bcl-x gene contains G4s that influence alternative splice site choices and identified one chemical compound (GQC-05) that promotes the formation of the minor pro-apoptotic isoform of Bcl-x both in vitro and in cells. This is of importance because this molecule could be further developed to induce cancer cell deaths.
To better understand the function and specificity of GQC-05 in alternative splicing, we propose to:

1. Determine the effects of GQC-05 and other G4 ligands on transcription and alternative splicing transcriptome-wide using RNA-seq experiments from cells incubated with various G4 ligands. We will gain information on their specificity of action, correlate the structural features of the ligands with their effects in cells and identify correlations with the distributions of potential G4-forming sequences.

2. Provide a molecular description of G4 formation and binding of GQC-05 in Bcl-x pre-mRNA. We will identify the G nucleotide involved in G4 formation in Bcl-x by a novel method based on phosphorothioate nucleotide analogue interference (NAIM) and confirm the contribution of these nucleotides in alternative splicing by specific chemical modifications. We will determine the structural basis of the specificity of GQC-05 to Bcl-x pre-mRNA G4 sequences.

3. Synthesize GQC-5 analogues with novel properties. We will define a structure-activity profile of GQC-05 analogues to improve their affinity to RNA and specificity in alternative splicing regulation.

4. Decipher the mechanisms by which G4s ligands affect splice site selection by investigating whether they either perturb the binding of splicing factors with critical roles in splice site selection or promote their recruitment using biochemical methods and TIRF single-molecule imaging.

Underpinning this research is our new technology for identifying functional G-quadruplexes (G4s) in pre-mRNA and our evidence that (a) G4s do affect splicing in Bcl-X, (b) the splicing patterns of Bcl-X and Mcl-1 respond strongly in vitro and in cells to only one of a wide range of G4 ligands, and (c) we have shown that this ligand binds and affects the structure of the RNA around the known G4-forming regions.

Both Bcl-X and Mcl-1 are important determinants of cancer cell survival and the best ligand shifts splicing strongly to the pro-apoptotic (death) patterns. We will investigate this process in detail (the first mechanistic analysis of the actions of G4s on splicing) and use this knowledge to design, synthesize and test a suite of biologically active compounds and specific tools in collaboration with GlaxoSmithKline (GSK).

1. Potential Economic Impact of the Research
Therapeutics & tool compounds for biotechnology and pharmaceuticals: new opportunities for the development of a new class of splice-switching small molecules and probes of splicing; new methods of controlling gene expression by creating RNA motifs that provide ligand-inducible splicing switches; new routes for developing cancer drugs with higher selectivity targeting known sets of genes.
Outputs: Patent protection of intellectual property; exploration of licensees, develop new chemical biological methods for industrial applications with GSK.

Mechanisms of delivering Economic Impact: we will apply to the many funds, some administered by UoL and UoS, available for translational research (see PTI for details) and seek alliances with GSK via an EPSRC industrial CASE studentship and with CRT.

2. People and Skills Development

PDRA1 will learn RNA biochemistry (very rare in the UK) and transcriptomics. PDRA2 will learn a wide range of skills in structural biology of RNA, under-represented in the UK, and single molecule methods available only in ICE's laboratory. The synthetic PDRA3 will develop skills in reaction optimization and photo-crosslinking using dedicated high-throughput facilities in Dr Jacob Bush's laboratory. All three PDRAs will be mentored, appraised annually and disseminate their findings at major conferences.

Outputs: Enhanced industrial-academic collaboration and upskilling of post-doctoral researchers; International exposure to academic excellence by presenting at conferences.

3. Potential Societal Impact of the Research

The aim of this research is to understand how G-quadruplexes (G4s) and small molecules that bind to them (G4 stabilizers) influence gene expression by altering the outcome of RNA alternative splicing. Public engagement will enable broader appreciation of the importance of chemical biology approaches. Cancer - see above.

Outputs: Public discussion of the role of Chemical Biology in bioscience (Glasgow Science Centre; Glasgow Science Festival). Communication of our research in specific publications aimed at the general public as well as stakeholders and policy makers such as Horizon2020 portal.
Mechanisms of delivering Societal Impact: Delivery of presentations at the Glasgow Science Festival and Glasgow Science Centre events; dissemination of results through the mainstream press on UoS and UoL websites; industrial-facing workshops and symposia via GSK.

4. Academic impact

PDRAs will present their findings at national and international conferences, and SET for Britain in order to promote our findings to the scientific community and to the broader public.

Summary of Resources for the Delivery of Impact-Related Activities

i. one-day workshop and two-day symposium, attendance at conferences,
ii. travel costs associated with collaborative meetings,
iii. industrial engagement (one-day symposium)
iv. costs associated with 3 month secondment of PDRA3 to GSK.