Award details

Drosophila Down Syndrome Cell Adhesion Molecule: A paradigm for revealing hidden splicing codes

Reference	BB/T003936/1
Principal Investigator / Supervisor	Dr Matthias Soller
Co-Investigators / Co-Supervisors	Dr Roland Arnold
Institution	University of Birmingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	513,009
Status	Current
Type	Research Grant
Start date	01/03/2021
End date	30/11/2025
Duration	57 months

Abstract

In almost every human gene the coding sequence can be varied by alternative splicing (AS) of the pre-mRNA to generate molecular and cellular diversity from a limited number of genes. Defects in AS are a major cause of human disease including metabolic disorders, cancer and neurodegeneration. AS also changes during aging. Principally, splicing defects caused by aberrant use of splice sites can be corrected, but requires thorough understanding of AS regulation. AS occurs with high accuracy even though the sequences involved are very degenerate relying only on two strictly conserved nucleotides on either end of an intron. Hence, a major obstacle in understanding the regulation of AS is our lack in recognizing regulatory elements involved in recruiting spliceosomal components to correct splice sites flanking an intron. Once splice sites are determined, the megadalton spliceosome forms and the intron is removed in a two step process involving formation of a lariat to the branchpoint adenosine generally close to the 3'splice site. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is a classic text book example for AS. Here, extraordinary molecular diversity is generated through mutually exclusive AS from four variable clusters to generate 36'016 different protein isoforms, yet we do not understand how AS in this model gene is regulated. We have developed a toolkit in a Drosophila model to answer the key questions regarding AS regulation of Dscam. Using a combination of reporter gene analysis and bioinformatic sequence analysis we will test a) whether remote branchpoints prevent splicing together of variable exons, b) whether long-range base-pairings direct choice of Dscam variable exons, and c) whether each variable cluster employs a unique code consisting of distinct regulatory elements to regionalize AS. Overall these studies will uncover novel mechanisms of AS regulation important for biological functions relevant to ageing and human health.

Summary

The exciting prospect of exploiting genome information for personalized medicine critically depends on the extent to which we understand the regulatory information residing outside the protein-coding regions of the genome. A unique feature of genes in eukaryotic organisms is their organization into protein-coding DNA sequences, termed exons, which are separated by non-coding introns. During splicing, introns are excised from the pre-messenger RNA (mRNA) transcript by the spliceosome and exons are joined to form the mature mRNA. A functional protein can then be made from the mRNA, but only if splicing controlled by hundreds of proteins has accurately taken place. The unique organization of eukaryotic "genes in pieces" further allows exons to be included in one mRNA from a particular gene, but excluded in another. This process, termed alternative splicing (AS), is used in most human genes and is an important mechanism to build complex organisms with comparatively few genes. AS is particularly prevalent in the brain and changes during aging. Mis-regulation of AS is also associated with various human diseases, including cancer, metabolic disorders and neurodegeneration. Fidelity of splicing rests critically on accurate reading of 'splicing information' in non-coding regions of the pre-mRNA. Paradoxically, introns are often very large and contain numerous sequence motifs that look like splice sites. Hence, the splicing information is encrypted in a code of short sequence motifs that we do not understand very well. As the splicing process is very complex, it is also vulnerable to cause human disease from mutations present in our genomes that result in aberrant splicing. In fact, about 15% of genetic human disease is caused by mutations in splice sites, but considering all regulatory elements involved in splicing, estimates range up to 50%. However, a drug consisting of a short stretch of nucleotides has recently been approved in the US and the EU for correction of splicing in the Spinal Muscular Atrophy (SMA) gene. Since such drugs can be directed to any part in the genome, many cases of aberrant splicing causing human disease could potentially be corrected. To make full use of this technology we need to understand the splicing code. The fruit fly Drosophila has proven an excellent and cost-effective genetic model for deducing basic biological processes as illustrated by the 2017 Nobel prize award. To discover fundamental splicing codes the Down Syndrome Cell Adhesion Molecule (Dscam) is an excellent model gene, because it is extensively alternatively spliced in four arrays of variable exons where only one exon is chosen for inclusion in the mature mRNA. This way, 36'016 different protein isoforms can be generated, which are more proteins from one gene than genes are present in the genome. This diversity is essential for development of the brain, but also in the immune system for recognition and clearance of pathogens such as bacteria. The most central questions regarding Dscam AS is why exons in the variable clusters are not spliced together despite having consensus splice sites and how one variable exon is chosen. We now have developed a toolkit in the fruit fly Drosophila that allows us to test a) whether splicing together of variable exons is prevented by splicing signals being too close together to allow for assembly of a functional splicesome, b) whether long-range base-pairings are key to Dscam AS to bring a variable exon into the proximity of flanking constant exons and c) whether each variable cluster contains unique regulatory sequences that restrict splicing to specific parts of a gene. From these experiments we will learn about fundamental mechanism involved in AS regulation and how their mis-regulation can lead to human disease. Our results will be instrumental for elucidating the splicing code to instruct how human disease caused by splicing errors can be corrected.

Impact Summary

DISEASE AND HEALTH CARE: Defects in mRNA splicing are a major cause of human genetic disease. The prospect to treat splicing defects with anti-sense oligo nucleotides or U7 snRNA based anti-sense effectors offers highly promising avenues for therapeutic interference to correct aberrant splicing. Genome information of individual patients will soon be available for diagnostic purposes and personalized therapies in our health care system. However, for most of the non-coding regions of the genome we are as yet unable to assign regulatory functions. Regulatory sequences involved in alternative mRNA processing, due to their degeneracy and redundancy, typically resist in silico determination of which proteins bind to them or which RNA-based processes they regulate and thus require experimental determination. In order for health care professionals to exploit genome information more fully, it is imperative to understand mRNA processing codes among non-coding sequences that frequently differ between individuals. Our research will provide major contributions to decipher mRNA processing codes related to alternative mRNA splicing. Alternative mRNA splicing changes as individuals age and better understanding of this process promises the opportunity for improved diagnostics and potentially new therapeutics leading to major enhancements in this segment of life. In the long-term identifying the splicing code promises to implement anti-sense technology as broad therapeutic approach to interfere with alternative mRNA splicing in a gene-specific fashion. We will actively pursue the exploitation of new data that might potentially underpin future patents. Thus, this proposal is directly relevant to the demand for trained scientists experienced in interpreting genome data. Indeed we will equip trainees with the requisite genetic and bioinformatics experience to accelerate advances in this crucial area of research. BBSRC STRATEGIC PRIORITIES: This proposal encompasses the BBSRC's strategic plan to enhance understanding of the most complex human organ, the brain. Since alternative mRNA splicing is abundant in the brain and is altered during aging, the proposed project provides a route for elucidating mechanisms that are risk factors for poor physical and mental health. This project is of particular relevance to the 10-year vision of the BBSRC towards an integrative understanding of organisms as it aims to elucidate regulatory codes at a genomic scale and how these codes operate at an organismal level. This project also fulfils a strategic aim: "developing and embedding a Systems approach to Biosciences in order to advance fundamental understanding of complex biological processes". Research on alternative mRNA splicing has been strongly focused on integrating information from diverse experimental situations. Thus, this project fulfils a BBSRC research priority of "Systems approach to biological research" and implements aims towards predictive biology. TRAINING: "Providing skilled researchers needed for academic research" is part of BBSRC Strategic aims: We have excellent track records in training and mentoring students and postdoctoral trainees at all levels and provide significant input in laboratory training for individual students as part of University degree programs. This includes graduate students participating in the UoB BBSRC funded doctoral training programs. This grant proposal promises to help BBSRC achieve this aim, especially as it will disseminate cutting edge technology and high-end quality science such as combining genetic with bioinformatic analysis for the interpretation of genomes to set standards for the youngest generation entering careers in research.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search