Multimerisation of ELAV/Hu proteins - a key mechanism ensuring fidelity of alternative splicing regulation

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

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 organisation into protein-coding DNA sequences, termed exons, which are separated by non-coding introns. During splicing, introns are excised from the pre-mRNA transcript by the spliceosome and exons are joined to form the mature messenger RNA (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, is used in most human genes and is an important mechanism to build complex organisms with comparatively few genes. Alternative splicing is particularly prevalent in the brain and changes during aging. Misregulation of alternative splicing is also associated with various human diseases, including cancer and neurodegeneration.
Fidelity of splicing rests critically on accurate reading of 'splicing information' in non-coding regions of the pre-mRNA. The splicing information is encrypted in a code of short sequence motifs that we do not understand very well. Paradoxically, genes that are spliced differently appear to have similar regulatory sequences. Evidently, evolution has generated a strategy to decrypt such splicing information, but it is upon us now to decipher this code. Knowing the splicing code will allow us to interpret genome sequences of regulatory regions, which are the sequences differing most among individuals.
The splicing code is read by RNA binding proteins shaped complementary to short parts on the RNA surface. Imagine a gecko, whose toes tightly attach to the slightly uneven surface of a wall. Only the combinatorial use of all its toes allow it to run up the wall. Accordingly, a concept has emerged implementing combinatorial binding of RNA binding proteins for generating an extended surface to bind to RNA and thereby providing specificity in RNA recognition and alternative splicing regulation. To date, however, little is known how RNA binding proteins assemble to provide this level of specificity.

To understand this novel mechanism in alternative splicing regulation, work in our laboratory has focused on ELAV (Embryonic Lethal Abnormal Visual system) proteins originally identified in Drosophila, consisting of a family of highly related proteins with homologues in humans called Hu proteins. ELAV/Hu proteins are prototype RNA binding proteins containing three RNA Recognition Motifs (RRM) and are predominantly expressed in neurons. An inherent property of ELAV/Hu proteins is their ability to multimerize. Hence, ELAV/Hu proteins represent an ideal system to determine the structural framework of how multiple copies of RNA binding proteins adopt a complementary shape to RNA for gene-specifically regulating alternative splicing.
We have recently obtained a 3D tetramer structure of ELAV RRM3, the main multimerization domain, allowing now to dissect multimerization and RNA binding functions that reside in different parts of the structure. We therefore propose to a) determine the biochemical and biophysical properties leading to multimerization, b) determine how multimerization contributes to gene-specific alternative splicing regulation using Drosophila transgenes and c) how ELAV connects with core pre-mRNA processing machinery.
From these experiments we will learn about fundamental principles involved in alternative splicing regulation and how their misregulation can lead, in the case of ELAV/Hu proteins, to neurological disease. Our results will be instrumental for elucidating the splicing code and its interpretation by RNA binding proteins during aging.

Technical Summary

Alternative splicing (AS) generates enormous molecular and cellular diversity from a limited number of genes in eukaryotes. Defects in AS are a major cause of human disease and aberrant regulation of AS results in phenotypes associated with the aging brain, including synapse retardation, neuronal and retinal degeneration, as well as locomotion deficits.
Regulation of gene-specific AS is governed by short sequence motifs, variably spaced in non-coding regions of the pre-mRNA. Paradoxically, genes that are spliced differently appear to have similar regulatory sequences. It has been proposed that combinatorial binding of RNA-binding proteins is the fundamental principle to ensure specificity in AS regulation. However, this concept has not yet been systematically investigated. A promising strategy to address this challenging question is to investigate AS regulation by ELAV/Hu proteins, which we and others have previously demonstrated to form defined multimers. ELAV/Hu constitutes a family of evolutionarily highly conserved RNA-binding proteins containing three RNA-recognition motifs (RRM). The C-terminal RRM (RRM3) is essential for multimerisation of ELAV/Hu proteins, is involved in RNA-binding and has been implicated in coupling of AS to transcription.
We have very recently determined crystal structures of ELAV RRM3, displaying a tetrameric assembly, corresponding to its assembly state in sedimentation velocity experiments. Based on this novel structural information, our multi-disciplinary platform provides a unique opportunity to systematically dissect a) the structural determinants governing ELAV multimerisation, b) the role of ELAV/Hu multimerisation in regulating AS of specific target genes using Drosophila transgenes, and c) whether interaction with co-regulators of AS is dependent on ELAV/Hu multimerisation.
Overall these studies will uncover novel mechanisms of AS regulation important for biological functions relevant to ageing and human health.

Planned Impact

General biological and clinical context
The introduction of genome information of individual patients for diagnostic purposes and personalised therapies in our health care system appears to be a realistic prospect over the next 5 - 10 years. However, for most of the non-coding regions of the genome we are as yet unable to assign regulatory functions. In order for health care professionals to exploit genome information more fully, it is imperative to decipher regulatory codes in introns and untranslated regions. Establishing the principles of how regulatory proteins, including those of the ELAV/Hu family, read cognate RNA sequences in non-coding regions plays a critical part in this endeavour, especially since comparative sequence analysis of the relevant sequence space has limited effectiveness in the face of massive degeneracy and redundancy.

Significance of ELAV/Hu proteins in potential clinical applications
More than 600 research papers in the past 5 years illustrate the scientific prominence of ELAV/HU proteins in the context of pre-mRNA processing. Importantly, clinical relevance and potential for therapeutic intervention is beginning to emerge. For instance, it was shown recently that micro-RNA based suppression of the human ELAV homologue HuR was able to suppress tumour growth in lung, ovary and kidney tissues (Abdelmohsen K, et al., Cell Cycle 9(7) (2010)). Also, downregulation of HuR was linked to doxorubin resistance in breast cancer cells (Latorre et al. (2012), Mol Cancer 11:13). These examples underscore the importance of RNA-processing in tumorigenesis and tumour progression, while highlighting the potential for conceiving novel approaches to interfere with gene-specific alternative RNA processing for therapeutic purposes. Although our study focuses on the Drosophila protein, the high level of sequence conservation to the human homologues and the mechanistic parallels between these proteins strongly suggest that research outcomes from our study will be relevant to the function and behavior of the human orthologues.

Engagement and interactions of applicants with stakeholders
Although research in the applicant's lab is basic research, it will instruct applications in cancer treatment and in areas of drug development and safety. MS has made contact with clinicians in the Thorax Clinic of the West Midlands Hospital to explore applications for the outcome of basic research about ELAV/Hu proteins for novel leads to treat highly therapy resistant Small Cell Lung Cancer, that ectopically express human ELAV proteins. In addition, ELAV proteins require tight regulation in the brain as either reduced or increased levels result in neurological defects. MS has made contact with Industry, in particularly the Toxicology Unit of AstraZeneca to implement Drosophila models for assessing low level chronic exposure of xenobiotics for neurotoxic effects mediated through ELAV proteins.

Engagement with general audiences
KF has been actively engaged in interacting with the general public and school children over a number of years. In 2005, he contributed centrally to preparing and presenting a science exhibition - Research Showcase - at this University, that involved organising exhibits, writing promotional material as well as speaking to visitors on the exhibition days. In addition, KF has been a frequent visitor to Schools in the Midlands as ambassador for the School of Biosciences. He has also been involved in outreach efforts to Biology departments of local secondary schools, and is regularly involved in Open Day activities.
MS has joined this University only relatively recently, and thus has not yet had the opportunity to engage with the public in an extensive fashion. Recently, MS has been involved in Open Day
activities, presenting research talks accessible to a general audience and in presentations for cancer biologists and clinicians.
 
Description This project is ongoing in studying how ELAV RNA binding proteins use multimerization to increase target specificity. As part of the molecular genetic analysis of ELAV/Hu protein function, we discovered that concentrations and cellular localization of the three ELAV proteins are important determinants for target specificity in Drosophila. Honeybees in contrast have only one ELAV protein and we discovered that honeybees use extensive alternative splicing to make up for the three ELAV genes in Drosophila, but alternative splicing in ELAV is not affected by neonicotinoids.
To study the function of RNA binding proteins sophisticated transgenic tools are required to control expression of these regulators, but also adequate reporter genes. We thus developed novel recombinant DNA and transgenesis technology to study the functions of these RNA binding proteins, in particular the role of multimerization in providing splicing fidelity. These tools will allow for defining the splicing code and for predicting how genes are regulated based on sequence variation present between individuals. We further discovered by generating mutations in the multimerization domain of ELAV that multimerization of ELAV is essential for regulation of alternative splicing in transgenic animals. This finding has fundamental impact on how recognition of RNA targets is evaluated for generating specificity in a cellular environment.
We further have now developed the technology to generate mutations in the endogenous locus and have now obtained mutants defective in multimerization, which are currently analysed.
Exploitation Route Our findings have important implications for defining the splicing code and for predicting how genes are regulated based on sequence. Differences between humans are mostly found in non-coding regions where RNA binding proteins bind. Hence such sequence polymorphisms will affect how genes are expressed. In the future, drugs can be developed to modulate the binding of proteins to RNA and adjust gene expression to improve human well being.
Further, we have developed novel technology for the manipulation of large DNA constructs to assess regulation of gene expression in transgenic Drosophila, but applicable to a wide range of model organisms.
We have discovered that honeybees use extensive alternative splicing to make up for the three ELAV genes in Drosophila, but alternative splicing in ELAV is not affected by neonicotinoids.
Sectors Healthcare

 
Description Responsive mode project grant
Amount £438,440 (GBP)
Funding ID BB/K006827/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2013 
End 02/2017
 
Description Open day showcase of Drosophila research 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact open day presentation of research using a Drosophila model to understand genetic disease
Year(s) Of Engagement Activity 2022
 
Description Out rearch at the Birmingham Think Tank at vrious events, e.g. brain awarness week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact At this event use of Drosophila for neuroscience research was displayed using microscopes and sample brains illustrating how brain research is facilitated by a genetic model organism.
Year(s) Of Engagement Activity 2022,2023,2024
 
Description Research presentation: Use of genetically modified model organsims for research on pest management 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We presented our research on pest management at the Birmingham Bee keeper association organized honey show at Martineau Gardens. There was a general interest in the research done. Explaining our research and discussions on the topic raised acceptance of genetically modified organisms.
Year(s) Of Engagement Activity 2022
 
Description Talk at the Birmingham Bee Keeper Association meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact At this talk at the Birmingham Bee Keeper Association meeting on research using Drosophila and honeybees 40 people attended. There were vivid discussions on how research contributes to understanding of toxicity of various agrochemicals used.
Year(s) Of Engagement Activity 2022