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PROTEOMICS New Tool Speeds Study of Mammalian Protein FunctionMethod May Provide Quicker Alternative to Knockout Animal Models In a study released in the April 16 Proceedings of the National Academy of Sciences, HMS researchers report a new vector that can "silence" the production of specific proteins in mammalian cells. The method has potential as a tool to speed the investigation of protein function--a field opened up by the sequencing of the human and other genomes. And it may eventually be used as a platform for developing new therapies.
Members of the Yang Shi lab have developed a new vector that silences gene expression in mammalian cells without having to first go through a time-consuming manipulation of germ line tissue. Christina Soohoo, El Bachir Affar, Yang Shi, Frédérique Gay, Yujiang Shi, and Guangchao Sui (l to r) believe the new technology will find broad application in genomics and medicine. (Photo by Jeff Cleary) The technology was developed by researchers in the laboratory of Yang Shi, HMS associate professor of pathology and principal author. The availability of the new method extends the range of possibilities of selective interference of gene expression without having to manipulate DNA in the egg or embryo. The vector-based technology makes it possible to turn off genes in a highly specific manner, and its effect is persistent unlike earlier RNA interference methods for human cells. Previously, to study mammalian protein function, many researchers had to go the laborious route of making knockout animals, a process that can take many months. The new technology complements knockout techniques and enables researchers to conduct a variety of experiments with cells grown in vitro, many of which represent differentiated cell types often difficult to obtain as primary cultures. Worm EnvyThe success of Shi's experiments has depended on recent advances in understanding RNA interference (RNAi), in which double-stranded RNA triggers the specific degradation of corresponding messenger RNA. This process, which leads to gene silencing, is observed in worms, insects, plants, zebrafish, and protozoa. Cells guard their genomes against viruses with RNAi, and, as studies in C. elegans have shown, it is likely important in developmental timing. Phillip Sharp of MIT pointed out in a recent review that this antiviral system is ancient and ubiquitous, with origins predating the plant-animal divide. The interplay between RNAi and transposons has, he said, "most certainly shaped the genomic structure of most organisms." For years researchers studied diverse manifestations of RNA interference--post-transcriptional gene silencing in virally infected plants and "quelling" in fungi--without grasping the underlying molecular connection. Yet this did not prevent them from provoking interference by applying exogenous RNA and using this as a powerful tool to knock out gene function, even though its workings remained mysterious. "Genes could be silenced in C. elegans by soaking them in RNA," said Shi, "or even by feeding them RNA-loaded bacteria." A simplistic model for RNA action was that the antisense strand bound messenger RNA and blocked translation. But that did not explain the "curious" observation that both sense and antisense strands appeared to be equally effective. Then researchers Andrew Fire and Craig Mello discovered that double-stranded RNA, sometimes present in almost homeopathic concentrations, was actually the active agent. "In retrospect it was obvious," said Shi. "It was staring right at us." When double-stranded RNA was injected into the gonad of the worm, the protein expressed by the target gene was not made in the offspring. In mammalian systems this would be the equivalent of making a knockout mouse with the prick of a needle. "People were envious of C. elegans researchers because they could do RNA interference," Shi said. Though RNAi had been reported in mouse oocytes, advances in mammalian systems were not forthcoming. Generally, applying double-stranded RNA to mammalian cells triggered a wide-ranging, interferon-mediated antiviral response. Translation stalled, and cellular ribonucleases degraded mRNA without discrimination. Then, in 1999, double-stranded RNAs that were 25 nucleotides in length were isolated from virally infected plant tissue. And it was shown that a component in Drosophila cell extracts chopped double-stranded RNA into fragments of equally small size. It became clear that these small interfering RNAs mediate gene silencing. Though the details of the process may differ among organisms, a general model has emerged: double-stranded RNA is the necessary substrate that is chopped up by the endonuclease DICER into nonoverlapping snippets. The antisense strand of these small bits binds mRNA, primes production of new double-stranded RNA synthesis, and so feeds the cycle. The potential for amplification of this natural process, sometimes called "degradative PCR," explains the potency and self-sustaining nature of RNA interference. It was this conceptual advance that set the stage for key experiments in Germany in 2001. In what Shi called a "groundbreaking" discovery, researchers in Thomas Tuschl's lab synthesized double-stranded RNAs that were 21 nucleotides in length. Applied to mammalian cells in tissue culture, these smaller RNA fragments were able to slip by cellular monitors. Finally, mammalian biochemists had achieved gene-specific silencing. The VectorsBut synthetic RNA has its limitations. The vector system devised by the Shi lab (and a similar vector reported online in the March 21 Sciencexpress) represent the next step. The salient features of the vector include a promoter for human RNA polymerase III, flanked on the 3' end by a termination signal composed of five T residues. Between start and stop signals is a 21-nucleotide DNA template and its inverted repeat separated by a short non-homologous nucleotide spacer. Transcription was predicted to produce a 21-nucleotide double-stranded RNA joined at a hairpin loop. In addition to circumventing the need for laboratory synthesis of double-stranded RNA fragments, an advantage of the RNA-silencing vector is that it induces persistent silencing of protein synthesis, whereas the response to synthetic RNAs is transient. "There are experiments that just can't be easily done with synthetic double-stranded RNA under those conditions," Shi said. Bill Forrester, HMS assistant professor of pathology, and members of the Shi lab, Guangchao Sui, Christina Soohoo, El Bachir Affar, Frédérique Gay, and Yujiang Shi, constructed a series of these vectors, each containing unique DNA sequences from a different endogenous gene. In each case, when the plasmid was transferred to cultured cells, small interfering RNAs were made, and there was robust inhibition of specific target protein synthesis. The silenced endogenous genes represented diverse functional protein classes: from housekeeping to cell cycle control to DNA methylation. Furthermore, transfections were done in HeLa cells, non-small cell lung carcinoma, HPV-negative cervical carcinoma, and osteosarcoma. Regardless of cell type, target genes were silenced, demonstrating the utility of the vector. Shi believes the vector may have direct medical application. Some forms of acute leukemia arise following chromosomal translocation in which one normal gene is spliced to another. The resulting aberrant protein is oncogenic. He hopes that small interfering RNAs to target the gene-gene junction might specifically knock down oncogene expression. "There's going to be a lot of interest in this technology," Shi said. --Anne Mahon |
