What is splicing and alternative splicing?

When a gene is switched on, the DNA sequence is copied into a molecule of messenger RNA (mRNA) which is then translated in a protein. The many thousands of genes in an organism generate proteins with different functions which either carry out chemical reactions or are building blocks for cells and organs. The majority of protein-coding genes in plants and animals contain introns which must be removed from precursor mRNAs to produce mRNAs. This process is called splicing and involves conserved splicing signals in the intron and the assembly of a large complex of around 200 proteins. It has been estimated that at any moment in time, each cell in the human body is engaged in splicing 20,000 introns.

Figure 1: Alternative splicing of pre-mRNA gives rise to different mRNAs and proteins.

Figure 1: Alternative splicing of pre-mRNA gives rise to different mRNAs and proteins.

Alternative splicing is where more than one mRNA is produced from the same precursor messenger RNA (pre-mRNA), giving rise to functionally different proteins (Figure 1). In humans there are approximately 30,000 genes and it is estimated that ~95% of human protein-coding genes undergo alternative splicing to generate up to 150,000-200,000 mRNAs and proteins through alternative splice site usage.

The importance of splicing and alternative splicing is highlighted by the number of human diseases which are associated with mistakes in the splicing process. These include muscular dystrophy, cystic fibrosis, Parkinson’s and many cancers.

Is alternative splicing prevalent in plants?

Plant introns differ from those of vertebrates and yeast by being in general smaller and U-rich. While the recognition of introns in the pre-mRNA is likely to differ, the basic mechanisms of spliceosome assembly and removal are expected to be similar. As in animals, splicing and alternative splicing is an important level at which gene expression is regulated.

Currently more than 60% of plant genes are known to be alternatively spliced and this is highly likely to be an underestimate. As alternative splicing generates protein complexity and can regulate mRNA levels via turnover of transcripts, it is extremely important in the control of gene expression in most, if not all, developmental and biochemical processes in the plant.

Key questions in alternative splicing in plants are what are the cis sequences and the trans-acting protein factors which determine splice site selection, how do the relative levels of factors in different cells (the ‘cellular code’ determine patterns of alternative splicing in different genes and is alternative splicing of genes involved in the same pathway co-ordinated?

To address these questions, we study splicing factors which are regulators of alternative splicing, have developed a high- resolution alternative splicing RT-PCR panel to monitor hundreds of alternative splicing events simultaneously, are developing process-specific alternative splicing panels and other systems to follow alternative splicing. In addition, we are collaborating with the two main European labs involved in alternative splicing research in plants: Andrea Barta (Vienna) and Artur Jarmolowski (Poznan) and are part of the European Alternative Splicing Network of Excellence – EURASNET.

Why is alternative splicing important in plants?

Among the genes where alternative splicing has been described are genes involved in development, disease resistance, biochemical and physiological pathways, cell division and chromosome integrity and include many transcription factors and RNA binding proteins. In particular, alternatively spliced genes are abundant among genes which are up- or down-regulated in stress conditions (for example, cold, heat, drought, high salt). Using the high-reslotion RT-PCR panel, we have identified numerous significant changes in alternative splicing under stress conditions suggesting that alternative splicing is sensitive to stress and may represent a rapid response to such conditions.