RNA

V

Other categories of RNA

Messenger, ribosomal, and transfer RNA are the main kinds of RNA in living cells. Mention was also made of small nuclear RNA (see above, under mRNA). The cytoplasm contains a further class of RNA, called small cytoplasmic RNA (scRNA), which mainly exists in the form of RNA-protein complexes. Within a special region of the nucleus, called the nucleolus, another class of RNA is found. It is called small nucleolar RNA (SnoRNA) and functions in the manufacture of ribosomal RNA. In addition, and of great recent importance, is the discovery, and recognition of the role, of small RNA molecules that function to control gene expression.

VI

Ribozymes

Biologists had traditionally thought that all enzymes (the molecules that catalyse the metabolic processes of life) are proteins. This view was overturned in the 1980s, when Thomas Cech of Yale University discovered that some RNA molecules can also act as enzymes. RNA molecules that act as enzymes are called ribozymes.

The Group I introns were the first ribozymes to be discovered. They catalyse their own excision from the mRNA; that is, they ‘self-splice’. Genes, particularly in eukaryotes, are made up of exons and introns (see DNA). The exons code for amino acids and are ultimately translated into a protein. The introns are sequences of nucleotides within the coding region and are spliced out of the mRNA (see above, messenger RNA). The most important kind of splicing occurs in the nucleus of eukaryotic cells. However, introns are also found in organellar genes, in mitochondria for instance, and even some bacterial and viral genes. These non-nuclear gene introns mainly belong to a particular category of introns called Group I introns. (Group I introns are also found in some nuclear genes, such as in the single-celled ciliates; but they are absent from nuclear genes in large, complex eukaryotes such as worms, flowers—see Angiosperm—flies, and humans.)

Group I introns are not removed at a special spliceosome. They remove themselves. The intron lies between two exons. It can transfer the chemical bone between itself and the end of the next upstream exon, such that the upstream exon bonds directly to the next downstream exon. The chemical reaction is called trans-esterification. As the trans-esterification occurs, the intron cuts itself out of the mRNA. No protein catalyses this reaction. After it was discovered that Group I introns could self-splice, it was found that Group I introns could catalyse other reactions in test-tube conditions. All the reactions involved trans-esterification.

RNA molecules are now being found to catalyse a rapidly expanding list of biological activities. The bacterium Escherichia coli contains an enzyme called ribonuclease P. Ribonuclease P contains both a proteins and an RNA molecule. It is an endonuclease: it can split an RNA chain, creating two shorter RNA molecules from one longer RNA molecule. Traditionally, it was thought that the protein part of ribonuclease P acted as the enzyme. However, it was experimentally shown that, under certain conditions, the protein part is unnecessary. The RNA part alone can catalyse the RNA-splitting reaction. In fact the RNA part of ribonuclease P directly catalyses the splitting, and the protein part accelerates the reaction under living conditions.

VII

Antisense RNA and RNA Interference (RNAi)

During the transcription of DNA to produce a polypeptide chain, a single strand of mRNA is produced from double-stranded DNA. With the exception of the replacement of thiamine (T) residues with uracil, the mRNA that is produced by this process is the same sequence as one of the strands of the DNA molecule—the “sense” strand. In fact, the RNA polymerase enzyme “reads” the other strand of DNA (the “antisense” strand) to produce a copy of the sense strand through complementary base pairing. Therefore, most mRNA within most cells is a copy of the sense strand of the DNA, which is subsequently used (through the process of translation) to produce polypeptides. Experiments aimed at controlling gene expression through the use of antisense RNA sequences indirectly led to the discovery of RNA interference (RNAi). It was originally postulated that if an RNA molecule were produced in a cell that was complementary to the sequence of a specific mRNA, then double-stranded RNA could form that would inhibit the ability of the mRNA to function during translation. That is, the formation of a double-stranded RNA molecule should inhibit the production of the specific protein encoded by the mRNA. This was indeed found to be the case, but the mechanism of reduction in gene expression does not proceed through the inhibition of translation, but via RNA degradation. Antisense technology was exploited commercially in the FlavrSavr® tomato. In 1994, the US Food and Drug Administration granted the first licence for human consumption to a genetically modified food. The FlavrSavr® tomato was modified so that it produced an antisense version of the polygalacturonase gene. The expression of the antisense RNA reduced the production of polygalacturonase mRNA. The enzyme encoded by this mRNA is involved in the breakdown of the fruit as it ages. The inhibition of the function of this enzyme led to the formation of tomatoes that could be grown to full ripeness on the vine prior to picking and transportation. Unmodified tomatoes have to be picked before they are ripe so that they are not damaged during transportation. The tomatoes are then ripened close to the point of sale using ethylene gas.

Some other observations did not fully explain how the phenomenon of gene regulation by antisense RNA occurred. For example, attempts by plant scientists to produce petunias with darker pigmented flowers through the insertion of additional copies of the pigmentation gene (encoding the protein chalcone synthase) into the plant found that plants that contain extra copies of the gene produced less pigmented, or even white, flowers. Further analysis indicated that the transcription of both the wild-type copy of the chalcone synthase gene and the additional copies of the gene added to the cell were reduced. In 1998, Andrew Z. Fire, Craig C. Mello, and colleagues published results that reconciled these and other apparently disparate observations. Fire and Mello were studying the expression of unc-22—a muscle protein—in the roundworm Caenorhabditis elegans. They found that injecting either unc-22 mRNA or antisense RNA into the worm had no effect on unc-22 protein levels. However, when they injected unc-22 double-stranded RNA, they observed gene silencing and a dramatic decrease in specific protein production. The two coined this phenomenon RNA interference, or RNAi, and were jointly awarded the Nobel Prize in Physiology or Medicine for their work in 2006.

The mechanism of action of RNAi is complex and still not fully understood. The RNAi pathway begins when an enzyme called dicer cleaves double-stranded RNA molecules into short fragments of approximately 20-25 base pairs in length. Dicer is an endo-ribonuclease that cleaves RNA to specific lengths and often cleaves a 2 base-pair overhang at the 3' end. The short RNA molecules produced by dicer are called small interfering RNA (or siRNA). One of these short RNA strands (the so-called guide strand) is then incorporated into a multi-protein complex termed the RNA-induced silencing complex (RISC). The catalytic component of RISC is a protein called argonaute. Argonaute binds to siRNA strands and then possesses endonuclease activity against the mRNA that the guide siRNA strand is complementary to. The remaining strand of the siRNA (the anti-guide strand) is also degraded by the RISC complex.

The natural role of RNAi seems to have evolved to protect cells against transposons (DNA segments that move) and viruses that may produce double-stranded RNA during their replication process. There are many examples of double-stranded RNA that occur naturally within cells, for example as part of the splicesome. These do not, however, induce RNAi, perhaps because this double-stranded RNA is also associated with cellular proteins. In plants, RNA silencing may be used as defence mechanism against viral infections. Viruses containing RNA genomes are strong inducers of RNA silencing since double-stranded RNA is formed during replication. Additionally, RNAi may confer immunity against closely related viruses. A mobile silencing signal (possibly double-stranded RNA) can spread from cell to cell in the plant to provide viral immunity. Some viruses are thought to circumvent RNA silencing by spreading very rapidly throughout the plant.

VIII

RNA as a molecule of biological inheritance

Most life on Earth uses DNA as the molecule of inheritance. RNA acts to implement the instructions in the DNA code. In DNA-based life, the enzyme that builds RNA (RNA polymerase) does so from DNA codes: it is DNA-dependent RNA polymerase. DNA-based life lacks an RNA-dependent RNA polymerase that would allow RNA to be copied directly and act as a molecule of inheritance.

However, the way that RNA carries information is essentially the same as in DNA and there is no reason in principle why RNA should not act as a hereditary molecule. In some life forms today, it does—in the RNA viruses.

RNA viruses consist of a short stretch of RNA codes, wrapped in a protein coat. Influenza virus and human immunodeficiency virus (HIV) are the best-known examples. The viruses can invade cells (human cells in these two examples) and use the cell's machinery to copy themselves. The exact mechanism by which RNA viruses copy themselves varies from one class of viruses to another. One large class of RNA viruses, including influenza virus, contain a gene for an RNA-dependent RNA polymerase. They can have themselves copied directly.

Another class of RNA viruses, called retroviruses, have a gene for reverse transcriptase. HIV is a retrovirus. Reverse transcriptase is RNA-based DNA-polymerase; it makes a DNA copy of the RNA in the virus. The DNA form of the virus is then used to run off multiple copies of the RNA virus, by normal transcription. Normal cells of DNA-based life forms lack reverse transcriptase and are unable to manufacture DNA from RNA master copy. Indeed the central dogma of molecular biology states that information flows from DNA to RNA to protein, and not in reverse. Retroviruses, which can achieve reverse copying from RNA to DNA, provide a small exception to the central dogma.

IX

The RNA World

RNA viruses are one real example in which RNA is a hereditary molecule. The second example is hypothetical. Early life, soon after the origin of life, may have used RNA rather than DNA for inheritance. Life on Earth originated about 4,000 million years ago, and fossils resembling modern (DNA-based) bacteria are known from 3,500 million years ago. The RNA world, if it existed, would probably have been in the time interval between 4,000-3,500 million years ago. DNA is unlikely to have been the original molecule of inheritance because it contains many features that only work in relatively advanced life forms. RNA could work in simple life forms: it can carry information in a single strand and interact directly with the environment.

RNA can act as a ribozyme to catalyse biological processes. (DNA cannot, because it always takes the form of a non-reactive double helix, except when it has been unwound.) If life took the form of an RNA world near the origin of life, ribozymes would have had to catalyse RNA replication. (RNA is now copied by protein enzymes, but protein manufacture is an advanced process that would probably have been absent in the earliest life forms.) Some ribozymes are known that can catalyse the replication of other RNA molecules, for instance by assisting in the bonding of an A nucleotide to a U nucleotide in the other molecule. However, no one has yet discovered an RNA that can act as a ribozyme to catalyse the replication of itself. If found, such an RNA would be the simplest possible self-replicating system. It would resemble the kind of molecule that many biologists imagine to have been ancestral to all life on Earth. The failure so far to discover such an RNA may mean that RNA in fact cannot self-replicate. If so, either RNA-based life may have been preceded by some other form of life or the whole hypothesis of an RNA world may be invalid. Alternatively, it may only be a matter of time before a ribozymal self-copying RNA-polymerase is discovered.

Critics of the RNA world hypothesis have pointed to various properties of RNA that cast doubt on whether it could have been the hereditary molecule early in life. For instance, RNA is unstable at medium and high temperatures, and the conditions under which life arose on the early Earth are thought to include high temperature. However, the RNA world remains a popular hypothesis about early life. If the hypothesis is correct, then modern DNA-based life, in which DNA is the hereditary material, most enzymes are proteins, and RNA is mainly an intermediary between DNA and protein, is descended from ancestors in which DNA and proteins were absent and the RNA itself both catalysed the metabolic reactions and acted as the hereditary molecule.