New approach to identifying different products of a single gene

21 November 2008

In humans, animals and other higher eukaryotes, a single gene may generate a variety of different messenger RNA (mRNA) products, and subsequently alternative forms of protein with different functions. The mechanism whereby this occurs is referred to as alternative splicing. Usually a single gene is not arranged in a contiguous stretch but is spread out in physically separate sections across the DNA strand. During alternative splicing, the precursor mRNA is separated and reconnected (with deletions and/or different combinations of exons) in order to produce varying mRNA products (splice variants).

It has previously been estimated that 74% of genes undergo alternative splicing and are able to produce different mRNA products, but the true extent of alternative splicing was thought to be greater than this. Although alternative splicing has been studied for sometime, methods such as microarrays and expressed sequence tags (ESTs) that had been used to identify splice variants were not able to detect closely related isoforms of mRNA (very similar but non-identical mRNAs) or differences in isoform levels. Now, using high-throughput sequencing, two surveys published in Nature and Nature Genetics have shown that splicing occurs in up to 94% of human genes. This technique not only allows identification of different splice variants of a single gene, but also the levels at which they are expressed, thereby allowing more detailed identification of gene products.

In both studies an inventory of gene and mRNA isoform expression was produced by deep sequencing the complementary DNA (cDNA) produced from mRNA. In their study Wang et al. analysed mRNA isoform expression in 10 different tissues and 5 cell lines, and showed that different splice variants are expressed in different tissues  [Wang ET et al. Nature (2008) doi: 10.1038/nature07509]. This was also shown by Pan et al. in the seven tissues they examined [Pan Q et al. Nat. Genet. (2008) doi:10.1038/ng.259]. Wang et al also investigated if there was variation in the isoform expressed in the same tissue - in this case the cerebral cortex - between individuals. They showed that although there is some variation between individuals in the splice variants they express, this was not as much as the variation observed between different tissues from the same individual. The next step involves determining the functions of the different splice variants and what regulates their expression in different tissues. In addition, this technique can be utilised to study isoform switching (which occurs in cancer cells), which may lead to better understanding of the disease and improved therapeutics.


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