February 05, 2003
RNA Interference Speeds Discovery Of Purposes Of Genes

RNA-mediated interference (RNAi) is being used as a technique to more easily turn genes off in order to discover their purposes. Caenorhabditis elegans (or C. elegans) is a perfect organism to use for RNAi experiments.

A quirk of the physiology of C. elegans means that such gene inactivation can occur simply if the RNAi molecule is eaten by the worm. And luckily for the researchers, the preferred diet of this little worm is the bug that for decades has been used in thousands of lab experiments - the bacterium E coli. Simply inserting the RNAi sequences into E coli and allowing the worms to feed resulted in the chosen gene being knocked out.

The technique is remarkably fast. "It used to take a year to knock out a gene, now with RNAi one person can knock-out every gene in just a few months," says Ahringer.

To support this work the scientists had to develop a way to grow bacteria strains that each contained the ability to make a different RNAi aimed to knock out a different target gene.

"The worms eat the bacteria ... silencing the gene in the worm and her progeny," Julie Ahringer, of the Wellcome Trust/Cancer Research UK Institute of Cancer and Developmental Biology at the University of Cambridge in England, told UPI. "We optimized this ... technique and then worked out methods to efficiently engineer the large number of bacterial strains needed (one for each gene)."

Since it is so easy to deliver RNAi molecules into C. elegans its being used for experiments to rapidly discover what many genes do. This has sped up experiments that rely on knocking out specific genes by orders of magnitude. Recently the use of RNA interference led to the discovery 400 genes in C. elegans worm that affect fat storage.

Scientists at Massachusetts General Hospital (MGH) and their colleagues have scoured thousands of genes in the C. elegans worm and have come up with hundreds of promising candidates that may determine how fat is stored and used in a variety of animals. The findings, published in the Jan. 16 issue of Nature, represent the first survey of an entire genome for all genes that regulate fat storage.

The research team led by Gary Ruvkun, PhD, of the MGH Department of Molecular Biology, and postdoctoral fellow Kaveh Ashrafi, PhD, identified about 400 genes encompassing a wide range of biochemical activities that control fat storage. These studies were conducted using the tiny roundworm Caenorhabditis elegans, an organism that shares many genes with humans and has helped researchers gain insights into diseases as diverse as cancer, diabetes, and Alzheimer's disease.

Many of the fat regulatory genes identified in this study have counterparts in humans and other mammals. "This study is a major step in pinpointing fat regulators in the human genome," says Ruvkun, who is a professor of Genetics at Harvard Medical School. "Of the estimated 30,000 human genes, our study highlights about 100 genes as likely to play key roles in regulation of fat levels," he continued. Most of these human genes had not previously been predicted to regulate fat storage. This prediction will be tested as obese people are surveyed for mutations in the genes highlighted by this systematic study of fat in worms.

In addition, this study points to new potential therapies for obesity. Inactivation of about 300 worm genes causes worms to store much less fat than normal. Several of the human counterparts of these genes encode proteins that are attractive for the development of drugs. Thus, the researchers suggest that some of the genes identified could point the way for designing drugs to treat obesity and its associated diseases such as diabetes.

Of the 400 genes which RNAi-based screening identified to affect fat metabolism about half have known human counterparts.

To discover this treasure trove of fat regulators, the researchers inactivated genes one at a time and looked for increased or decreased fat content in the worms. Through this time-consuming process, they identified about 300 worm genes that, when inactivated, cause reduced body fat and about 100 genes that cause increased fat storage when turned off. The identified genes were very diverse and included both the expected genes involved in fat and cholesterol metabolism as well as new candidates, some that are expected to function in the central nervous system.

About 200 of the 400 fat regulatory worm genes have counterparts in the human genome. "A number of these worm genes are related to mammalian genes that had already been shown to be important in body weight regulation. But more importantly, we identified many new worm fat regulatory genes, and we believe that their human counterparts will play key roles in human fat regulation as well," says lead author Ashrafi. "The work was done in worms because you can study genetics faster in worms than in other animal models, such as mice," says Ashrafi. "The model is a great tool for discovering genes."

RNAi allowed the relevant genes to be identified out of a much larger set of genes.

The work was dependent on the use of an RNA-mediated interference (RNAi) library constructed by the MGH team's collaborators at the Wellcome/Cancer Research Institute in England. The library consists of individual genetic components that each disrupt the expression of one particular gene. With this tool, the researchers were able to systematically screen almost 17,000 worm genes for their potential roles in fat storage.

Now that the bacteria have been created that make each type of RNAi for C. elegans many other effects of genes can be looked at. Already the original researchers have used this technique to look at genes that affect longevity.

In another paper, Dr. Ruvkun and Dr. Ahringer have used the RNA method to screen the worm's genome for genes that increase longevity. With two of the six chromosomes tested, they have found that genes in the mitochondria, the energy-producing structure, are particularly important in determining life span.

This result demonstrates how the use of RNAi can support massive rapid screening of a large number of genes in order to identify a relevant subset for a particular purpose. This is not the only such recent result of this nature.

RNAi is being used to control the expression of the gene for p53 which is a crucial protein for regulating cell proliferation. Mutations in areas of the genome that control p53 expression are known to be crucial in the development of some types of cancer.

The study showed that establishing different levels of p53 in B-cells by RNAi produces distinct forms of lymphoma. Similar to lymphomas that form in the absence of p53, lymphomas that formed in mice with low p53 levels developed rapidly (reaching terminal stage after 66 days, on average), infiltrated lung, liver, and spleen tissues, and showed little apoptosis or "programmed cell death."

In contrast, lymphomas that formed in mice with intermediate p53 levels developed less rapidly (reaching terminal stage after 95 days, on average), did not infiltrate lung, liver, or spleen tissues, and showed high levels of apoptosis. In mice with high B-cell p53 levels, lymphomas did not develop at an accelerated rate, and these mice did not experience decreased survival rates compared to control mice.

The study illustrates the ease with which RNAi "gene knockdowns" can be used to create a full range of mild to severe phenotypes (something that geneticists dream about), as well as the potential of RNAi in developing stem cell-based and other therapeutic strategies.

Along with a recent study by Hannon and his colleagues that demonstrated germline transmission of RNAi, the current study establishes RNAi as a convenient alternative to traditional, laborious, and less flexible homologous recombination-based gene knockout strategies for studying the effects of reduced gene expression in a wide variety of settings.

RNA interference will be used in a newly announced effort to look at 10,000 genes for signs that they play roles that would make them relevant in understanding the causes of cancer.

This has been made possible by the discovery of a process called RNA interference which is used by the body to switch off individual genes while leaving all others unaffected.

The charity Cancer Research UK and the Netherlands Cancer Institute plan to join forces to exploit this knowledge to inactivate almost 10,000 genes one at a time in order to find out precisely what they do - and how they might contribute to cancer's development.

RNAi is being used as a tool to study the effects of shutting down a gene that causes soybean allergies.

Last September, for example, Anthony J. Kinney, a crop genetics researcher at DuPont Experimental Station in Wilmington, Del., and his colleagues reported using a technique called RNA interference (RNAi) to silence the genes that encode p34, a protein responsible for causing 65 percent of all soybean allergies. RNAi exploits the mechanism that cells use to protect themselves against foreign genetic material; it causes a cell to destroy RNA transcribed from a given gene, effectively turning off the gene.

How the double-stranded RNA gets used by the cell to turn off genes.

When double-strand RNA is detected, an enzyme called dicer, discovered at the Cold Spring Harbor Laboratory on Long Island, chops the double-strand RNA into shorter pieces of about 21 to 23 bases. The pieces are known as small interfering RNAs or siRNAs. Each short segment attracts a phalanx of enzymes.

Together, they seek out messenger RNA that corresponds to the small RNA and destroy it. In plants and roundworms, the double-strand RNA can spread through the organism like a microscopic Paul Revere.

The cell's reaction to double-stranded RNA in this manner may have evolved as a defense mechanism against double-stranded RNA virus invaders.

This page has links to some published papers that involve working with RNAi.

RNA interference (RNAi) is the process where the introduction of double stranded RNA into a cell inhibits gene expression in a sequence dependent fashion. RNAi is seen in a number of organisms such as Drosophila, nematodes, fungi and plants, and is believed to be involved in anti-viral defence, modulation of transposon activity, and regulation of gene expression.

Share |      Randall Parker, 2003 February 05 12:59 AM  Biotech Advance Rates

Mr. Iannazzo said at April 5, 2003 10:55 AM:

If there are any researchers looking for a worker in there research, especially in RNA Inerteference
please contact me!

Daljit said at May 23, 2003 7:15 PM:

My dad has a brain tumor and is listed as terminal. If anybody knows of a clinic or insitution that is using RNA interference on humans, please let me know. Right now, he is using Careseng which is helping him feel better but I don't think that's enough. Thanks for any input you might have

Divya Raj said at September 12, 2004 7:23 PM:

If theres anyone who knows which institutes in India are working on RNAi in vivo models (preferably mice)please tell me.

my mail id:divyabrill@yahoo.co.in.

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