MicroRNA evolution put to the test

Two Nature Genetics papers probe evolution and functionality of microRNAs and their target sites

Oct 30, 2006
Jeffrey M. Perkel
MicroRNA genes and their target sites are under Darwinian selection and continue to evolve, according to a pair of papers out this week in Nature Genetics. Nikolaus Rajewsky used genotyping data to show that predicted microRNA target sites are under negative Darwinian pressure, while Ronald Plasterk used a massively parallel sequencing approach to identify several hundred candidate microRNAs, many of which are restricted to humans, primates, or vertebrates in general. "My reaction is, this is why we put the genomes out there," said Bob Waterston, chair and professor of genome sciences at the University of Washington, who helped generate many of the genome sequences used in the Plasterk paper. "You can see these papers taking advantage of the wealth of sequence information... I find it very gratifying." "It's an advance for evolutionary biologists and systems biologists who are interested in how humans are wired," added Phillip Zamore, Gretchen Stone Cook Professor of Biomedical Sciences at the University of Massachusetts Medical Center. "It gives them another variable for their models." MicroRNAs are short, non-coding RNAs that post-transcriptionally repress gene expression either by blocking translation or inducing the degradation of targeted mRNAs. Several hundred miRNAs and tens of thousands of potential targets have already been identified or predicted, but two basic questions remained: how many more miRNAs are there, and which of the predicted target sites are functional? "The whole issue of being able to both predict microRNAs and then to predict their targets is a big challenge," said Waterston. Plasterk, professor of developmental genetics at the University of Utrecht, the Netherlands, used massively parallel sequencing technology to produce 400,000 sequence reads from small RNAs found in human fetal brain and adult chimpanzee brain. After filtering out irrelevant reads such as tRNAs, rRNAs, and other known miRNAs, Plasterk's team was left with 244 novel human and 230 novel chimpanzee candidate miRNAs, most of which were expressed at very low levels. The team then used comparative genomics to examine the conservation of these transcripts over hundreds of millions of years of evolution. Eight percent of the novel human miRNAs identified in this study were restricted to humans, more than half were restricted to primates, 30% were limited to mammals, and 9% were restricted to nonmammalian vertebrates or invertebrates. In several cases, said Plasterk, the genomic location of the miRNA genes was found to be dynamic across evolutionary time; for instance, there might be a single gene at the human miRNA locus, but two genes at the corresponding chimp position. "I like to see that, because microRNAs could be an important factor in sculpting development in evolution," he said."The idea that microRNAs can contribute to species identity has been bandied about for some time, and this is nice confirmation of that," said Zamore. "We're beginning to home in on what makes us, us."In the second paper, Rajewsky, of the Max Delbruck Center for Molecular Medicine in Berlin, looked over a much shorter stretch of evolutionary time to determine whether predicted miRNA target sites are under negative Darwinian pressure. Rajewsky used single nucleotide polymorphism data from the HapMap and Perlegen genotyping projects, which effectively are limited to the time since humans radiated out of Africa, to analyze the allele frequencies of human polymorphisms in predicted miRNA binding sites from the 3' untranslated regions of mRNAs. Rajewsky found that SNP density and allele frequencies were indicative of negative selective pressure acting on these target sites. Importantly, the team observed such selective pressures both on sites that are evolutionarily conserved and on those that are restricted to humans. "That's very cool, because previously people thought those [non-conserved] sites might not be functional," said Zamore. "This new approach lets us estimate the number of predicted sites that do make a contribution to human fitness, not only for evolutionarily conserved predicted targets, but also for sites specific for humans," said Rajewsky. Based on his data, Rajewsky estimates that about 85% of conserved miRNA target sites are likely to be functional. In addition, "we showed that if microRNAs and their targets are coexpressed, then 30% to 50% of [nonconserved] predicted sites are likely to be functional," Rajewsky said. "That's kind of neat, because it is a population genetics technique applied to making statements about a whole layer of gene regulatory control and its evolution in humans."The negative pressure on these miRNA target sites suggests mutation in these sites could lead to disease, said Rajewsky. "We now have a couple hundred candidates for investigating human disease, because we found variations in sites that are presumed to be functional." Next, he will apply his technique to other non-coding, regulatory elements, such as transcription factor-binding sites. The net result of these two studies, according to Zamore, is a novel method for predicting and validating human-specific (that is, nonconserved) miRNA target sites. "If you have a microRNA that is unique to humans, how would you find its target computationally? All the really robust algorithms rely on conservation, so by definition they cannot be used to find targets of human-specific microRNAs," said Zamore. "Now you use the SNP data among humans from the Rajewsky method to look for negative selection against loss of microRNA target complementarity."Jeffrey Perkel jperkel@the-scientist.comLinks within this article:C. Choi, "MicroRNA target practice," The Scientist, June 20, 2005. http://www.the-scientist.com/article/display/15538Nikolaus Rajewsky http://www.mdc-berlin.de/englisch/about_the_mdc/public_relations/press_releases_2006/pr_2006.htm# Ronald Plasterk http://www.niob.knaw.nl/researchpages/plasterk/groupleader.htmlBob Waterston http://waterston.gs.washington.edu/Phillip Zamore http://www.umassmed.edu/bmp/faculty/zamore.cfm454 Life Sciences http://www.454.comE. Berezikov et al., "Diversity of microRNAs in human and chimpanzee brain," Nature Genetics, advance online publication, Oct. 29, 2006. doi: 10.1038/ng1914 http://www.nature.com/ng/journal/vaop/ncurrent/abs/ng1914.htmlK. Chen, N. Rajewsky, "Natural selection on human microRNA binding sites inferred from SNP data," Nature Genetics, advance online publication, Oct. 29, 2006. doi:10.1038/ng1910 http://www.nature.com/ng/journal/vaop/ncurrent/abs/ng1910.htmlA. Constans, "A practical guide to the HapMap," The Scientist, Feb. 1, 2006. http://www.the-scientist.com/article/display/23052Perlegen Sciences http://genome.perlegen.com