Projects

- Abstract:

January 2003 the Carlsberg Foundation granted 5 years of support for the project Molecular Diversity and Evolution. The grant includes 10 post doc years, 2 PhD stipends and running costs.

The project has two major components: 1. Evolution of processes responsible for gene product diversity. Focus will initially be on alternative splicing in evolution and 2. Evolution of regulatory networks based on non-coding RNA. The overall perspective for these studies is the molecular evolution of organism complexity. It is the intention better to understand the relation between phenotypic evolutionary change and the evolution of basic molecular processes. Methodologically, the project combines bioinformatics and molecular experimentation including the use of custom expression microarrays and nematodes as model organisms.

We specifically wish to focus on the evolution of splicing control and regulation and the involvement of non-coding RNA including antisense transcript regulation. Bioinformatic as well as experimental skills are preferable

The project is in close collaboration with John Mattick's group at the Institute for Molecular Biosciences at University of Queensland, Australia, the Bioinformatics Center, University of Copenhagen, the Department of Statistics and Operational Analysis, University of Copenhagen and the Danish biotech company Exiqon.

- Project description:

Molecular diversity and Evolution

A molecular evolution research project funded for five years (2003-2008) by the Carlsberg Foundation.

Introduction

Molecular genetic efforts still primarily concentrate on changes in the DNA. However, there is a strong emerging interest in which products that actually are expressed. This is one of the reasons for proteomic efforts to register all proteins in a given cell. This project focuses on the processes that lead to the protein diversity in the cells and, so to speak, places it self right between genomic and proteomics. Focus is on evolutionary studies of these molecular processes and how they integrate with the evolution of the organisms phenotype, thus analyzes are carried out in a phylogenetic context. It has not previously been possible to study the molecular diversity, we do not know how it evolves and the influence on the phenotypic changes we see.

Genes can be spliced in different ways according to developmental stage, environmental factors such as stress or because of malfunctioning as in many diseases. Whether the different splice patterns are highly conserved or on the contrary that new or modified patterns are flexible and play a significant role in organisms’ evolution, is unknown. It is not unthinkable that evolutionary changes in these processes can play a significant role in the evolution of complex organisms. The core issue becomes evolutionary changes in regulatory systems contrary to more traditional views.

For eukaryot organisms gene expression can be modified in the process from DNA to protein. Hundreds of different types of modifications are described, however the frequency and importance of them is unknown. Alternative pre-mRNA splicing have lately been shown to take place for at least half of the human and other complex eukaryot genes and differential splicing is probably more the rule than the exception.

The combination of different splice sites and RNA editing can potentially result in more than a million different gene products (Gravely, 2001). Thus, only a single gene and only two involved modifications can potentially express protein diversity significantly larger than the organisms’ total number of genes. The possible number of products stemming from an organisms DNA is unthinkably high and likewise is obviously the number of potential combinations. Alternative splicing in evolution is one of the two main projects.

Regulation of splicing is still uncovered. Pro- and eukaryote regulation systems are probably fundamentally different, and regulation of the extremely complex eukaryote genetic systems is likely to be based on network-like systems. In the last few years it has been shown that non-coding RNA genes (ncRNA, not translated into protein) play determining regulatory roles. Theoretically, ncRNA has the capacity to form the framework of eukaryot molecular and genetic systems (Mattick & Gagen, 2002). In order to open a window for understanding the evolution of regulatory systems and molecular diversity and complexity. The second project is a phylogenetically based, combined bioinformatic and experimental analysis of regulatory ncRNA.

Evolution of alternative splicing
Expression micro arrays have been chosen as the major technical methodology for this research as it gives access to whole cell/organism information. The nematode, Caenorhabditis elegans has been chosen as model organism because its biology is known in much detail, its genome is sequenced and it is relatively easy to grow. C. briggsae, a fairly close relative, has also recently been sequenced which has a great impact on experimental- as well as bioinformatic comparative studies. Both species are routinely grown at the Department of Evolutionary Biology.

A number of genes have been chosen based on different criteria for our first generation arrays. Elaborate control and reference probes system has been developed and we have now proven our specific concept of detection of differential splicing. Arrays are being hybridized with reverse transcribed RNA from different species and strains of worms in different developmental stages and exposed to different sorts of stress.

Micro arrays include intensive use of LNA, an artificial nucleotide, because of its higher binding efficiency (http://www.exiqon.com). Development of custom arrays happen in close collaboration with the biotech company Exiqon, owner of the LNA patent.

Non-coding RNA in regulatory evolution
The overall goal is to examine the possible role of ncRNA networks in regulatory systems.

Preliminary work will focus on bioinformatic identification of different classes of regulatory ncRNAs in sequenced genomes.

It is likely that micro array based analysis also will be applied in this project.

From its start the project is collaboration between Department of Evolutionary Biology, Bioinformatics Center at University of Copenhagen, Department of Statistics and Operations Research also at University of Copenhagen and Institute for Molecular Biosciences (John Mattick), Brisbane, Australia.

Perspectives
The methods and insights gained from this project will largely be independent of the species studied. Principles and methods will bee directly applicable to any pair or group of organisms; such as mouse-rat, human-chimpanzee, cow-sheep etc. The phylogenetic distances between these mentioned organisms are not preventive for informative comparative analyses across all the species.

It is likely, that evolutionary biology is confronting a test of central paradigms. Current evolutionary biology is based on Darwin’s concept of selection combined with Mendelian heredity. Selection is well understood; heredity on the other hand, is well described, but not very well understood. First with molecular genetic developments have is it become possible to approach heredity in a qualitatively different way.

27.01.2003

Graveley, B. R. (2001): Alternative splicing: increasing diversity in the proteomic world. Trends in Genetics, 17(2): 100-107. Mattick, J.S. & Gagen, M.J. (2001): The evolution of controlled multitasked gene networks: the role of introns and other non-coding RNAs in the development of complex organisms. Mol. Biol. Evol. 18(9): 1611-1630.

Specific projects:

- Detecting alternative splicing in C. elegans using custom expression arrays with LNA spiked capture probes.
(- See ‘Technical Supplement’ )
- Detection and quantification of small truncations and exon skipping by rtPCR based assays.
- Evolutionarily conserved sequences at splice junctions.
- Antisense transcripts in splice regulation.