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Between Genes and Proteins
After DNA, scientists face the complex world of RNA
By Lone Frank

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Explaining the complexity of human biology is a different challenge today than it was before the publication of the human genome sequence. The sequence contains 30,000 genes, but our cells require many more proteins to do the work of the body. If genes encode multiple proteins, then the architect of biological complexity may be RNA—the molecule that directs the production of proteins from DNA.

Danish researchers are using microarrays to investigate alternative splicing in the nematode C. elegans. View larger

"The great future challenge in genomics is to understand how gene diversity is regulated," says population geneticist Peter Arctander, of Copenhagen University's Institute of Zoology. Regulation of how and when genes are turned into proteins can occur at several levels, but Arctander believes RNA is by far the most important generator of complexity. This go-between molecule harbors an enormous potential for creating variation, and the Copenhagen group is planning one of the first systematic studies of how an organism regulates this flexibility. "We live in an RNA world," says Arctander.

RNA's role in assembling a diverse collection of proteins is a product of evolution. Unlike genes in bacteria, genes in plants and animals are not arranged as continuous DNA sequences but as coding 'exons' interspersed with non-coding 'introns.' This cassette-like structure makes it possible to transcribe one gene into several different products as each 'messenger RNA' is spliced together from combinations of exons and bits and pieces of introns.

RNA editing is more common than textbooks tell us

Last spring, Arctander and colleagues published data suggesting that alternative splicing occurs much more often than they anticipated. "Previous estimates were that around 20 percent of human genes are transcribed in more than one alternative variant, but our analyses put the number closer to 50 percent," explains Arctander, who found similar results for other higher organisms such as mice and nematode worms. The findings appeared in Nature Genetics.

Similar studies now underway suggest that the estimate of 50 percent may be conservative, says collaborator and molecular biologist Søren Schandorff, of Protana Inc. in Odense, Denmark. "Our approach is to turn the usual way of looking at alternative splicing upside down." The Danish team uses computer analyses to look first at transcribed mRNAs and then compare each to the corresponding gene. This is done using two databases, one containing short stretches of DNA called expressed sequence tags (ESTs), which are derived from processed mRNA, and a second containing genomic DNA sequences. The researchers have evidence that RNA editing is more common than textbooks tell us.

Arctander is now moving into the lab to investigate RNA regulation in the living organism. In a collaboration with molecular geneticist Sakari Kauppinen of Copenhagen-based Exiqon Inc., he is mounting a study using microarrays, or gene chips, to look at alternative splicing in the nematode C. elegans. The aim is to characterize how these genes are transcribed in different developmental stages as well as in response to a number of environmental stresses. "At this stage it is necessary to concentrate on the basics," says Kauppinen. "Researchers need to systematically annotate actual splice variants and develop reliable detection systems and methods for analyzing the data."

Tracking the way splice variants are ultimately turned into protein is a project for another day. Schandorff does not doubt that in the future information on RNA regulation will be the basis for studying the expression of genes and proteins. Theoretically, many genes in higher animals can generate an almost unlimited number of different mRNA transcripts and, ultimately, proteins.

‘We have barely scratched the surface of genetic complexity.’

A prime example is a new study of genes that control brain development in the fruit fly Drosophila melanogaster. A team of American researchers has reviewed calculations indicating that so-called neurexin genes can give rise to 35,000 different possible protein products just from alternative splicing. "If you add to that the possibilities for RNA editing, as well as the various modifications that cells can confer to proteins after translation, you potentially end up with millions of different gene products," says Arctander. In fact, studies of fly species that have evolved separately for millions of years show that the sequences of many alternative splice sites are strictly conserved, indicating that they are indeed used.

"Seen from an evolutionary perspective, the potential flexibility inherent in alternative splicing may be a great advantage in speeding up development," says Arctander. But, he adds, "the fact that individuals within a species—be it flies or humans—are remarkably alike suggests that variation is normally tightly regulated." Based on this, Kauppinen predicts that gene regulation is likely to play a significant role in disease. Although the focus of many researchers has been DNA sequence variations, "understanding alternative splicing and RNA editing may be equally important," he says.

Delving into the highly complex universe of gene regulation calls for new ways of thinking, and Arctander predicts that systems biology will incarnate the next generation of molecular genetics. "We have barely scratched the surface of genetic complexity," he says. "To gain a deeper understanding we must move beyond the classical approach of investigating isolated biological elements and develop methods for looking at whole cells and organisms in an integrated way."

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Croft, L. et al. ISIS, the intron information system, reveals the high frequency of alternative splicing in the human genome. Nat Genet 24, 340-341 (April 2000).
Graveley, B.R. Alternative splicing: increasing diversity in the proteomic world. Trends Genet 17, 100-107 (February 2001).

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