New RNA world

Print edition : December 01, 2006

Andrew Z. Fire and Craig C. Mello pose next to a statue of German scientist Paul Ehrlich after being awarded the Paul Ehrlich and Ludwig Damstaedter award in Frankfurt on March 14, seven months before they won the Nobel Prize. - MICHAEL PROBST/AP

The Nobel Prize for Medicine and Physiology is awarded for the discovery of a mechanism of gene silencing by double-stranded RNA.

THIS year's Nobel Prize in Physiology and Medicine, like that in Chemistry, is for the discovery of a mechanism relating to the flow and regulation of genetic information coded in DNA (deoxyribonucleic acid). This mechanism goes beyond the fundamental process known as the Central Dogma of biology, that is, the transcription of genetic information in the double-stranded DNA, to a specific species of single-stranded RNA known as the messenger RNA (mRNA). This is translated from RNA into a protein by ribosomes in the cytoplasm of a cell. Not very long ago, RNA was regarded as just a disposable copy of the more important DNA; it was considered a passive messenger for protein synthesis that was destroyed once this process was completed.

Not any longer. A new RNA world has been unravelled in which RNA is an active participant in the chemistry of life.

In 1998, Andrew Z. Fire of Stanford University and Craig C. Mello of the University of Massachusetts Medical School discovered that double-stranded RNA (dsRNA) triggers the suppression of activity in specific genes. This mechanism, called RNA interference (RNAi), is activated when the RNA molecule occurs as a double-stranded pair in the cell. It degrades those mRNA molecules that carry a genetic code identical to that of the dsRNA. The gene corresponding to the mRNA is thus "silenced" and the encoded protein not made.

The Nobel Prize has been given to Fire and Mello for their discovery of this mechanism of gene silencing by double-stranded RNA.

Since only one strand of DNA is typically copied into RNA, the resulting mRNA is normally a single strand. Until recently, dsRNA was studied mainly as a transient molecule in the replication of RNA viruses, an intermediary that signalled cells of lower organisms to mount an anti-viral response. In the past few years, however, dsRNA and the associated mechanism of RNAi have emerged as important regulators of gene expression in higher organisms, including plants, animals and humans. This RNAi-based regulation of gene expression is important both for the development of an organism and the functions of cells and tissues. RNAi protects against certain RNA virus infections.It also keeps "jumping genes", or the mobile elements of the DNA, called transposons, under control and thus ensures stability of the genome. RNAi is already being used as a method to study the function of genes and may lead to novel therapies in the future.

Around 1990, plant biologists observed some striking effects that were difficult to explain. In one experiment, they were trying to increase the colour intensity of the petals in petunias by introducing a cloned gene to induce the formation of red pigment in the flowers. But instead this led to a complete loss of colour and the petals turned white. The enigma was resolved after Fire and Mello discovered RNAi.

The two were investigating the regulation of gene expression in the nematode worm Caenorhabditis elegans. They found that injecting mRNA molecules encoded to produce a muscle protein had no effect on the worms. The genetic code in mRNA is described as being a `sense' sequence. Injecting an `antisense' sequence mRNA, namely RNA with sequence complimentary to mRNA that should pair with mRNA, also had no effect. But when `sense' and `antisense' mRNA were injected together, it was observed that the worm displayed peculiar twitching movements. They observed similar effect in worms that lacked a functioning gene for the muscle protein.

Injecting sense and antisense sequences together allowed the two to bind and form a double-stranded RNA. Fire and Mello hypothesised that such a dsRNA molecule silenced the gene carrying the same code. To test this, they injected dsRNAs containing genetic codes for several other worm proteins. In every experiment they found that dsRNA carrying a particular genetic code silenced the function corresponding to the gene with that code. The protein encoded by that gene was no longer formed (Fig. 2).

After a series of simple and elegant experiments, they concluded that these genes were "silenced" dsRNA, this interference was specific for the gene whose code matched that of the injected RNA molecule, and other mRNAs remained unaffected. They further concluded the following: (i) the silencing was triggered efficiently by dsRNA, but weakly or not at all by single-stranded sense or antisense RNAs; (ii) the targeted mRNA disappeared suggesting that dsRNA not only silenced the RNA but also degraded it ; (iii) a few dsRNA molecules per cell were sufficient to achieve complete silencing, and therefore dsRNAs acted catalytically; and, (iv) the dsRNA effect could be transmitted between cells and could thus spread between tissues and even be inherited.

These results were published in their landmark paper in Nature on February 19, 1998, wherein they also suggested that RNAi offered an explanation for various unexplained phenomena observed in plants for several years. In a subsequent paper in the Proceedings of the National Academy of Sciences (PNAS), Fire also provided evidence to show that the degradation of the targeted mRNA occurred prior to translation; that is, the effect of the dsRNA was "post-transcriptional". He also provided a model for the catalytic function of the dsRNA in targeting mRNAs with matching codes.

Thus, the discovery of Fire and Mello clarified many confusing and contradictory experimental findings and revealed a new natural regulatory mechanism in gene expression. This heralded the beginning of a new research field. Within a year, the presence of RNAi was documented in many other organisms, including fruit flies, trypanosomes, plants, hydra and zebrafish. Initially, it was found that mammalian cultured cells did not display a potent and specific RNAi effect, apparently because of a non-specific physiological response of long dsRNAs. But when the cells were exposed to short, 21-nucleotide-long dsRNAs, an efficient and targeted silencing effect was seen in mammalian cells as well. Thus the generality of RNAi among eukaryotes was soon established with the exception, remarkably so, of the unicellular budding yeast, Saccharomyces cerevisiae, in contrast to the system serving as a perfect model for the DNA-mRNA transcription process.

The components of the molecular machinery underlying RNAi were discovered in the following years (Fig. 3) in an in vitro system using cultured cells of the fruitfly Drosophila melanogaster. It was demonstrated that dsRNA binds to a protein complex called Dicer, which cleaves it into fragments of short RNAs. Another protein, RISC (RNA-induced silencing complex) binds these fragments and, while one of the RNA strands is destroyed, the other remains bound to RISC. This serves as a probe to detect mRNA molecules. When an mRNA molecule with sequence complementary to the RISC-bound RNA is detected, it binds to the RISC complex and is cleaved and degraded. The gene corresponding to that code is silenced and no protein is synthesised.

The discovery of RNAi has far-reaching consequences given the under<147,2,7>lying molecular mechanism (Fig. 4). The discovery that cells can process injected dsRNA and eliminate homologous (matching) single-stranded RNA suggested that RNAi could provide a defence mechanism against viral attacks. Many viruses have a genetic code that contains dsRNA. When such a virus infects a cell, it injects its RNA molecule, which immediately binds to the Dicer molecule. RISC gets activated and the viral RNA gets degraded. Today, it has been established that this anti-viral mechanism is at work in plants, worms and flies. It is still unclear how relevant it is for mammals, including humans. However, higher organisms have an efficient immune system involving antibodies, killer cells and interferons.

The RNAi mechanism also provides genomic stability. DNA has short sequences that can move around the genome, called `jumping genes' or transposons. These are present in the DNA of all organisms and can cause damage if they end up in the wrong place, producing undesirable effects and even unwanted mutations. Transposons copy their DNA to RNA, which is reverse-transcribed and translocated at another site in the genome. Part of this RNA molecule is often double-stranded and can be targeted by RNAi. Though the mechanism is not yet understood fully, it is clear that if the RNAi machinery is not efficient, transposons are not kept under control and they can jump across the genome, causing deleterious effects. It has also been suggested that RNA silencing could be a kind of immune defence of the genome. Close to 50 per cent of our genome consists of viral and transposon elements that have invaded the human genome in the course of evolution. In a manner similar to the vertebrate immune system, the RNAi machinery can recognise invading dsRNA, mount a response and suppress infection by degrading the viral RNA.

Soon after the discovery that RNAi is effected at the molecular level through short RNAs (as a result of fragmentation by Dicer), it was shown that there is a class of genes - hundreds of them, in fact - in the genome that encode small RNA molecules called microRNAs. This is a feature shared by worms, flies, mice and humans. They contain pieces of the code of other genes. Such microRNAs form a double-stranded structure by base-pairing with mRNA and activate the RNAi machinery to block protein synthesis. The expression of that particular gene is silenced.

The significance of genetic regulation by microRNAs in the development of the organism and the control of its cellular functions is becoming clear. It is estimated that there are about 500 microRNAs in mammalian cells and about 30 per cent of all genes are regulated by microRNAs. It is now known that microRNAs play an important role in the development of plants, C. elegans and mammals. Thus, microRNA-dependent regulation of gene expression, that follows from RNAi, represents a major new principle. The discovery of microRNAs is regarded as major, though the full significance of regulation by these small RNAs is yet to be revealed.

The targeted effect of RNAi offers a new experimental tool in biology and biomedicine to repress specific genes and reveal the resulting phenotypic effect. The work of Fire and Mello on C. elegans has shown that the approach could be used on cells from almost all organisms, including mammalian cells. This new technique is now being exploited not only in cultured cells but also in transgenic organisms.

RNAi's likely impact on technology and health care, though perhaps not immediate, could become important. The concept opens up new possibilities in the use of gene technology and gene therapy. Double-stranded RNA molecules have been designed to activate the silencing of specific genes in humans, animals and plants. Such molecules are introduced into the cell to activate the RNAi machinery and break down mRNA with homologous code. In a recent work, a gene causing cholesterol was silenced in treating animals through this method. Plans are under way to develop silencing RNA as a treatment for viral infections, cardiovascular diseases, cancer, endocrine disorders and several other diseases.

But these are early stages. "The most immediate benefit," points out Fire, "is going to be in doing experiments that teach us things. I think the successes are going to be there but it is also going to take a little while and a lot of stamina from the people doing work. There may be things that don't work quite so well, there may be setbacks, as for any new therapeutic."

In 1989, Sidney Altman and Thomas Cech got the Nobel Prize for discovering that RNA can act as a catalyst. It was soon shown that RNA could catalyse its own replication and synthesis of other RNA molecules, much like the DNA itself. This changed our perception about RNA and led to the idea that RNA was the first genetic material on the earth.

An "RNA world" is believed to have existed before DNA took over as the key genetic material and RNA was relegated to the role of a messenger. The dramatic new knowledge of gene regulation by RNAi has further widened our perspective of RNA's active role. The implications of the discovery of RNAi to the evolutionary process could, in fact, be revelatory.

"I am very excited now," says Mello, "in thinking in terms of the role of this type of silencing in evolutionary change, rather than simply as something involved in regulating gene expression. Actually, I think it has a potential impact on evolution on a broader scale as a source of inheritance and variation."

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