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Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message. (See mechanism of translation.)
However, RNA can form duplexes just as DNA does. All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand; e.g.,
5´ C A U G 3´ mRNAThe second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense. When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked. This may occur because
3´ G U A C 5´ Antisense RNA
With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer.
Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe. However, these tomatoes often became damaged during shipment and handling and have been taken off the market.
|Right: Flower of a tobacco plant carrying a transgene whose transcript is antisense to one of the mRNAs needed for normal flower pigmentation. Left: Flower of another transgenic plant that failed to have its normal pigmentation altered. (Courtesy of van der Krol, et. al., from Nature 333:866, 1988.)|
In this respect, it is easier to produce transgenic plants than transgenic animals.
|Read more about the potential uses of transgenic plants at this LINK.|
Do cells contain genes that are naturally translated into antisense RNA molecules capable of blocking the translation of other genes in the cell? The answer is yes, and these seem to represent another method of regulating gene expression.
In both mice and humans, the gene for the insulin-like growth factor 2 receptor (Igf2r) that is inherited from the father synthesizes an antisense RNA that appears to block synthesis of the mRNA for Igf2r. An inherited difference in the expression of a gene depending on whether it is inherited from the mother or the father is called genomic or parental imprinting.
|Imprinting of the Igf2r gene.|
In testing the effects of antisense RNA, one should use sense RNA of the same coding region as a control. Surprisingly, preparations of sense RNA often turn out to be as effective an inhibitor as antisense RNA.
Why? It seems that the preparations of sense RNA often are contaminated with hybrids: sense and antisense strands that form a double helix of double-stranded RNA (dsRNA). Double-stranded RNA corresponding to a particular gene is a powerful suppressant of that gene. In fact, the suppressive effect of antisense RNA probably also depends on its ability to form dsRNA (using the corresponding mRNA as a template).
The ability of dsRNA to suppress the expression of a gene corresponding to its own sequence is called RNA interference (RNAi). It is also called post-transcriptional gene silencing or PTGS.
The only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded RNA. If the cell finds molecules of double-stranded RNA (dsRNA), it uses an enzyme called Dicer to cut them into small fragments. In plants 3 lengths are formed:
In each case, the two strands of each fragment separate — releasing the antisense strand. With the aid of a protein, both 21-nt and 22-nt molecules bind to a complementary sense sequence on a molecule of mRNA. If the base-pairing is exact, the mRNA is destroyed (21-nt) or its translation inhibited (22-nt). In all three cases, the result is inhibition of gene expression.
Because of their action, these fragments of RNA have been named "small (or short) interfering RNA" (siRNA).
The complex of siRNA and protein is called the "RNA-induced silencing complex" (RISC).
There is growing evidence that siRNAs can also inhibit the transcription of genes
How these siRNAs — synthesized in the cytosol — gain access to the DNA in the nucleus is unknown.
Synthetic siRNA molecules that bind to gene promoters can — in the laboratory — repress transcription of that gene. The repression is mediated by methylation of the DNA in the promoter and, perhaps, methylation of histones in the vicinity.
There is a strain of rice (LGC-1) that produces abnormally low levels of proteins called glutelins. It turns out that of several glutelin genes found in rice
RNAi has been found to operate in such diverse organisms as plants, fungi, and animals such as Drosophila melanogaster, Caenorhabditis elegans, and even mice and the zebrafish. Such a universal cell response must have an important function. What could it be?
In any case, the discovery of RNAi adds a promising tool to the toolbox of molecular biologists. Introducing dsRNA corresponding to a particular gene will knock out the cell's own expression of that gene. (Feeding C. elegans on E. coli manufacturing the dsRNA will even do the trick.)
In the 24 March 2005 issue of Nature, Sönnichsen et al reported that they have injected dsRNAs corresponding to 20,326 of C. elegans's genes (98% of the total!) and monitored the effect of each on embryonic development from the completion of meiosis (following fertilization) through the second mitotic division that produces the 4-cell embryo.They found that at least 661 different genes altered some process during this period:
(Another thousand genes produced phenotypic effects that were seen at later stages of development.)
Because RNAi can be done in particular tissues at a chosen time, it often provides an advantage over conventional gene "knockouts" where the missing gene is carried in the germline and thus whose absence may kill the embryo before it can be studied.
|Link to discussion of knockout mice.|
Another Example: screening genes for their effect on drug sensitivity
Some other promising applications of RNAi
In mammalian cells
In mammalian cells, introducing dsRNA fragments only reduces gene expression temporarily. However, mammalian cells can be infected with a DNA vector that encodes an RNA molecule of 50–80 nucleotides called a "small hairpin RNA" (shRNA) containing a sequence corresponding to the gene that one wishes to suppress. As the shRNA is synthesized, dicer converts it into a typical siRNA molecule. Because the cell can continuously synthesize shRNA, the interference is long-lasting. In fact, with vectors that become integrated in the host genome, RNAi can be passed on to the descendants.
The 19 June 2003 issue of Nature reported on coffee plants that were engineered to express a transgene that makes siRNA that interferes — by RNAi — with the expression of a gene needed to make caffeine. So perhaps "decaf" coffee will one day no longer require the chemical removal of caffeine from coffee beans.
Monsanto has received approval to market transgenic corn (maize) that expresses a dsRNA corresponding to the sequence of an essential gene in the western corn rootworm, a devastating pest of the crop. After ingesting this dsRNA, the insect's own cells process it into an siRNA that targets the gene's mRNA for destruction and kills the worm in a few days.
In C. elegans, plants, and Neurospora, the introduction of a few molecules of dsRNA has a potent and long-lasting effect. In plants, the gene silencing spreads to adjacent cells (through plasmodesmata) and even to other parts of the plant (through the phloem). RNAi within a cell can continue after mitosis in the progeny of that cell. Triggering of RNAi in C. elegans can even pass through the germline into its descendants.
Such amplification of an initial trigger signal suggests a catalytic effect. It turns out that these organisms have RNA-dependent RNA polymerases (RdRPs) that uses the mRNA targeted by the initial antisense siRNA as a template for the synthesis of more siRNAs. Synthesis of these "secondary" siRNAs even occurs in adjacent regions of the mRNA. So not only can these secondary siRNAs target additional areas of the original mRNA, but they are potentially able to silence mRNAs of other genes that may carry the same sequence of nucleotides.This phenomenon, called "transitive RNAi",
Because its target is so specific, the possibility of using RNAi to shut down the expression of a single gene has created great excitement that a new class of therapeutic agents is on the horizon.
Recently (10 August 2018) the U. S. FDA approved its first siRNA drug. Patisiran (Onpattro®) will be used to treat the genetic disorder hereditary transthyretin-mediated amyloidosis.
Transthyretin is a serum protein synthesized in the liver which transports retinol (vitamin A) and thyroxin in the blood. Mutations in the gene (TTR) encoding transthyretin can produce a modified protein with a propensity to clump into aggregates causing amyloid deposits which damage nerves and other tissues.
The patisiran siRNA binds to the transthyretin messenger RNA (mRNA) interfering with its translation and thus the synthesis of the mutated version of transthyretin. Patients treated with patisiran showed marked improvement of their signs and symptoms. (However, they often needed to take vitamin A supplements.)
|Another approach: CRISPR-Cas9 technology shows promise of another way to block the production of defective transthyretin. In a trial of 6 volunteer patients, a single injection of a CRISPR-Cas9 construct knocked out the defective TTR gene and lowered the level of defective transthyretin in their blood.|
Patisiran is likely going to be the first of many siRNA-mediated therapies. Many clinical trials are currently underway exploring the use of siRNA molecules in the treatment of a wide variety of diseases. One example:
A siRNA that binds to the messenger RNA from which a protein designated PCSK9 is translated reduces the level of low-density lipoproteins (LDLs, "bad" cholesterol) in the circulation. PCSK9 is a serine protease which binds to the complex of LDL and its receptor (LDLR) promoting the degradation of the receptor within the cell. With fewer LDL receptors returning to the cell surface [View] to remove more LDLs, the level of LDLs in the blood rises. By reducing the synthesis of PCSK9 within the cell, the siRNA brings down the concentration of "bad" cholesterol in the blood potentially reducing the risk of heart attacks and other cardiovascular problems.
|Another approach: Antisense oligonucleotides (ASOs) are synthetic molecules that — because they, too, are antisense — also block mRNA translation. Several have been approved for human therapy. [Link]|
In C. elegans, successful development through its larval stages and on to the adult requires the presence of at least two "microRNAs" ("miRNAs") — single-stranded RNA molecules containing about 22 nucleotides and thus about the same size as siRNAs.
These small single-stranded transcripts are generated by the cleavage of larger precursors using the C. elegans version of Dicer.
They act by either destroying or inhibiting translation of several messenger RNAs in the worm (usually by binding to a region of complementary sequence in the 3' untranslated region [3'-UTR] of the mRNA).
The microRNAs (miRNAs) in C. elegans (which were first called "small temporal RNAs") turn out to be representatives of a large class of RNAs that are encoded by the organism's own genes.
While direct evidence of the function of many of these newly-discovered gene products remains to be discovered, they regulate gene expression by regulating messenger RNA (mRNA), either
|Link to a discussion of riboswitches — another mechanism for regulating the translation of mRNA.|
MicroRNAs have two traits ideally suited for this:
MicroRNAs regulate (repress) expression of genes in mammals as well. Genome analysis has revealed thousands of human genes whose transcripts (mRNAs) contain sequences to which one or more of our miRNAs might bind. Probably each miRNA can bind to as many as 200 different mRNA targets while each mRNA has binding sites for multiple miRNAs. Such a system provides many opportunities for coordinated mRNA translation.A study reported in Nature (Lim, et al., 433: 769, 17 Feb 2005) used DNA chip analysis to show that when a particular miRNA was expressed in HeLa cells,
Thus repression of gene expression by miRNAs appears to be a mechanism to ensure regulated and coordinated gene expression as cells differentiate along particular paths. For example, when zygote genes begin to be turned on in the zebrafish blastula, one of them encodes a miRNA that triggers the destruction of the maternal mRNAs that have been running things up to then.
So miRNAs may play as important role as transcription factors in regulating and coordinating the expression of multiple genes in a particular type of cell at particular times.
The ease with which miRNAs can be introduced into cells and their widespread effects on gene expression have given rise to hopes that they might be useful in controlling genetic disorders, e.g., cancer.To date, some laboratory studies have been quite promising.
In addition to protein transcription factors, eukaryotes use small RNA molecules to regulate gene expression — almost always by repressing it — so the phenomenon is called RNA silencing.
There are two sources of small RNA molecules:
Aside from their use as laboratory — and perhaps therapeutic — tools, small RNAs are clearly essential to the organisms that make them.