| Index to this page |
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.
3´ G U A C 5´ Antisense RNA
This may occur because
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, enzymes synthesized by the tomato cause them to 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 spoilage. 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.
| Antisense oligodeoxynucleotides (ODNs) are synthetic molecules that - because they, too, are antisense - also block mRNA translation. One has been approved for human therapy. [Link] |
Do cells contain genes that are naturally translated into antisense RNA molecules capable of blocking the translation of other genes in the cell? Recently a few cases have been found 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 preparations of sense (and antisense) RNA often are contaminated with some hybrids; that is, the sense and antisense strands form a double helix of double-stranded RNA dsRNA. In any case, double-stranded RNA corresponding to a particular gene is a powerful suppressant of that gene.
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 mRNA. If the cell finds molecules of double-stranded RNA dsRNA, it uses an enzyme (the one in Drosophila has been named Dicer) to cut them into fragments containing 21-25 base pairs (~ 2 turns of a double helix).
The two strands of each fragment then separate enough to expose the antisense strand so that it can bind to the complementary sense sequence on a molecule of mRNA. This triggers cutting the mRNA in that region thus destroying its ability to be translated into a polypeptide.
RNAi has been found to operate in such diverse organisms as plants, fungi, and animals such as Drosophila, C. elegans, and even mice and the zebrafish. Such a universal cell response must have an important function. What could it be?
One possibility
Some viruses of both plants and animals have a genome of dsRNA. And many other viruses of both plants and animals have an RNA genome that in the host cell is briefly converted into dsRNA [link to examples]. So RNAi may be a weapon to counter infections by these viruses by destroying their mRNAs and thus blocking the synthesis of essential viral proteins.
Another possibility
In C. elegans, successful development through its larval stages and on to the adult requires the presence of at least two "small temporal RNAs" ("stRNAs")- single-stranded RNA molecules containing about 22 nucleotides - thus the same size as the fragments made by the Drosophila Dicer gene.
These small transcripts are generated by the cleavage of larger precursors using the C. elegans version of Dicer.
They act by inhibiting translation of several messenger RNAs in the worm (by binding to a region of complementary sequence in the 3' untranslated region [3'UTR] of the mRNA).
So RNA interference may be the unexpected dividend of a another basic process of controlling gene expression.
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.)
This can be done in particular tissues at a chosen time. This 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. |
| Welcome&Next Search |