RNAi: what's all the noise about gene silencing? - Focus

Environmental Health Perspectives, March 15, 2004 by Ernie Hood

It all began with petunias. In the late 1980s, geneticist Richard Jorgensen, then working at a California plant biotechnology company, attempted to deepen the hue of purple petunias by introducing more of the gene that gives them their color, in the form of double-stranded RNA (dsRNA). Instead, some of the engineered flowers became variegated and others turned white, indicating that expression of both the introduced pigmentation gene and its homologous endogenous gene had been knocked down or knocked out altogether. Jorgensen had serendipitously discovered an age-old natural biologic process now recognized to be evolutionarily conserved in most, if not all, forms of life. Today, gene silencing--or RNA interference (RNAi), as it is now known--has revolutionized genetics and is on the verge of spawning an entirely new class of drugs to treat human diseases with a genetic component.

The ability to selectively silence genes is one of the hottest topics in biology today. Science crowned RNAi as its "Breakthrough of the Year" in 2002. Nobel laureate and RNAi pioneer Phillip Sharp, who is Salvador E. Luria Professor of Biology and director of the McGovern Institute for Brain Research at the Massachusetts Institute of Technology (MIT), calls it "the most exciting discovery in the last decade," adding that "there's not an area of biological science this will not touch." John Maraganore, who is president, CEO, and director of Alnylam Pharmaceuticals, touts RNAi as "presenting perhaps the broadest new class of therapeutics since recombinant proteins and monoclonal antibodies."

Can RNAi live up to the hype? That remains to be seen, of course, but academic and industrial researchers are optimistic that it can and will, if the significant remaining barriers to its progress can be overcome. Given the rapid pace of discovery in the field, such optimism may well be justified.

RNA Redefined

It once seemed so simple, so straightforward: basically, DNA makes messenger RNA (mRNA); mRNA makes proteins. But the discoveries associated with RNAi have shown that the real story is far more complex. RNA has been unveiled as the "man behind the curtain" in the cell, wielding previously unimagined control over and influence upon cellular processes (including gene expression and regulation) and organism development. RNAi has been revealed to be an ancient mechanism protecting cells from invading viruses and from damage by transposable genetic elements, performing a variety of cellular housekeeping functions essential to survival, health, and development.

RNAi was first described and so named by molecular biologists Andrew Fire of the Carnegie Institute of Washington and Craig Mello of the University of Massachusetts, along with their colleagues, in a landmark 19 February 1998 Nature paper that electrified the biology community. The team found that administering tiny amounts of dsRNA to Caenorhabditis elegans resulted in potent sequence-specific gene silencing. Tantalized by the possibility of acquiring a powerful new tool for genetic manipulation and analysis, investigators around the world began investigating RNAi.

The flood of significant discoveries that followed soon established the basic outlines of the mechanisms involved in RNAi. Researchers using Drosophila found in 2000 that long-strand dsRNA was processed in cells into 21- to 23-nucleotide snippets of RNA, which then cleaved to precisely matching homologous mRNA sequences, degrading the mRNA and effectively silencing the corresponding gene by blocking its ability to encode for proteins. The higher life forms, such as mammals, while conserving this ability, use it in different ways; the response to dsRNA is more complicated, triggering a cellular immune response involving the release of interferon that ultimately kills the cell.

Then in 2001, Thomas Tuschl, then of the Max Planck Institute for Biophysical Chemistry in Gottingen. Germany, discovered with his colleagues that RNAi could be prompted through the use of shorter pieces of RNA, known as small interfering RNAs (siRNAs). Soon thereafter, they showed that duplexes of 21-nucleotide siRNAs mediated RNAi in cultured, mammalian cells and demonstrated that siRNAs could be designed to silence specific genes without activating the interferon response, in other words, scientists could potentially silence any gene of interest in a highly predictable, reproducible, and accurate fashion.

Research scientist Gregory Hannon and his colleagues at New York's Cold Spring Harbor Laboratory contributed several key discoveries during the same period. They identified, described, and named the "Dicer" enzyme, which chops dsRNA into siRNAs, as well as the RNA-induced silencing complex (RISC), which mediates the silencing process by degrading the homologous mRNA. In 2002, they described the use in mammalian cells of so-called short hairpin RNAs (shRNAs), which generate endogenous siRNAs within cells and thus provide stable, heritable gene silencing (in contrast, administered siRNAs are transient in their silencing effect). They whimsically named this effect "short hairpin-activated gene silencing," or SHAGging. This discovery allowed the development of cell lines and animal models with permanently silenced genes--a major step forward for basic science in general, and especially for functional genomics.