Unit Three. The Continuity of Life
Thus far we have discussed gene regulation entirely in terms of proteins that regulate the start of transcription by blocking or activating the “reading” of a particular gene by RNA polymerase. Within the last decade, however, it has become increasingly clear that RNA molecules can regulate the expression of genes, acting after transcription as a second level of control.
Discovery of RNA Interference
As will be discussed in chapter 13, the bulk of the eukaryotic genome is not translated into proteins. This finding was puzzling at first, but biologists now suspect that RNA transcripts of these regions might play an important role in gene regulation. The finding that almost all the differences between human and chimpanzee DNA occur in such regions only adds to the suspicion.
All this began to make sense in 1998, when a simple experiment was carried out, for which Americans Andrew Fire and Craig Mello later won the Nobel Prize in Physiology or Medicine in 2006. These investigators injected double-stranded RNA molecules into the nematode worm Caenorhabditis elegans. This resulted in the silencing of the gene whose sequence was complementary to the double-stranded RNA, and of no other gene. The investigators called this very specific effect gene silencing, or RNA interference. What is going on here? As you will learn in chapter 16, RNA viruses replicate themselves through double-stranded intermediates—at a critical stage, a virus enzyme called reverse transcriptase travels along the virus RNA and assembles a complementary strand. Because the life cycle of many viruses involves a double-stranded RNA stage, RNA interference may have evolved as a cellular defense mechanism against these viruses; the evolution of this adaptation would have predated the evolutionary divergence of plants and animals. Indeed, double-stranded viral RNAs can be targeted for destruction by RNA interference machinery. Without intending to do so, the nematode researchers had stumbled across this defense.
How RNA Interference Works
Investigating interference, researchers noted that in the process of silencing a gene, plants produced short RNA molecules (ranging in length from 21 to 28 nucleotides) that matched the gene being silenced. Earlier researchers were focusing on far larger messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) and had not noticed these far smaller bits, tossing them out during experiments. These small RNAs appeared to regulate the activity of specific genes.
Soon researchers found evidence of similar small RNAs in a wide range of other organisms. In the plant Arabidopsis thaliana, small RNAs seemed to be involved in the regulation of genes critical to early development, while in yeasts they were identified as the agents that silence genes in tightly packed regions of the genome. In the ciliated protozoan Tetrahymena thermophila, the loss of major blocks of DNA during development seems guided by small RNA molecules.
The first clue of how small fragments of RNA can act to regulate gene expression emerged when researchers noted that stretches of double-stranded RNA injected into C. elegans can dissociate. Each single strand can then form a double-stranded RNA by folding back in a hairpin loop, like the three sections of RNA shown toward the top of figure 12.19. This occurs because the two ends of the strand have a complementary nucleotide sequence. When the RNA loops, the complementary bases form base pairings that hold the strands together much as they do in the strands of a DNA duplex.
Figure 12.19. How RNA interference works.
Double-stranded RNA is cut by dicer. The resulting siRNA associates with proteins forming a complex called RISC. siRNA becomes single stranded and binds to targeted mRNAs with the same or similar sequences, which blocks translation of the gene.
Exactly how does such a double-stranded RNA inhibit the expression of the gene from which the double-stranded RNA has been generated? In the first stage of RNA interference, an enzyme called dicer recognizes long, double-stranded RNA molecules and cuts them into short, small RNA segments called siRNAs (small interfering RNAs) 1. In the next step, the siRNAs can assemble into a ribonucleoprotein complex called RISC (RNA Interference Silencing Complex) 2. RISC then unwinds the siRNA duplex, which leaves one single strand of RNA that is able to bind to mRNAs complementary to it 3 and thus silence the genes that produced those mRNA molecules.
Once the siRNA has bound to mRNA, the silencing is achieved in one of two ways: Either the mRNA is inhibited by blocking its translation into protein, or the mRNA is destroyed. The choice between inhibition and destruction is thought to be governed by how closely the sequence of the siRNA matches the mRNA sequence, with destruction being the outcome for best-matched targets.
Key Learning Outcome 12.8. Small interfering RNAs, called siRNAs, are formed from doublestranded sections of RNA molecules. These siRNAs bind to mRNA molecules in the cell and block their translation.
Biology and Stavina Healthy
Silencing Genes to Treat Disease
The recent discovery that eukaryotes control their genes by selectively "silencing” particular gene transcripts has electrified biologists, as it opens exciting possibilities for treating disease and infection. Many diseases are caused by the expression of one or more genes. AIDS, for example, requires the expression of several genes of the HIV virus. Many chronic human diseases result from excessively active genes. What if doctors could somehow shut these genes off?
The idea is simple. If you can isolate a gene involved in the disorder and determine its sequence, then in principle you could synthesize an RNA molecule with the sequence of the opposite or "anti-sense” strand. This RNA would thus have a sequence complementary to the messenger RNA produced by that gene. Introduced into cells, this synthesized RNA might be able to bind to the messenger RNA, creating a double-stranded RNA that could not be read by ribosomes. If an anti-sense therapy could be made to work and be delivered practically and inexpensively, the AIDS epidemic could be halted in its tracks. Indeed, any viral infection could be combatted in this way. Influenza is perhaps the greatest killer of all infectious diseases. A workable anti-sense therapy could provide a means of stamping out a bird flu epidemic before the virus spreads.
By far the most exciting promise of anti-sense gene silencing therapy is the possibility of practical cancer therapy. Discussed in chapter 8, cancer kills more Americans than any other disease. We now know in considerable detail how cancer comes about. It results from damage to genes that regulate the cell cycle. The great promise of RNA gene silencing therapy comes from those cancer-causing gene mutations that increase the effectiveness of one or more "divide” signals. If these mutant genes could be silenced, the cancer could be shut down.
The possibility of using complementary RNA to silence troublesome genes has gotten a huge boost in the last few years from the discovery of a unique virus defense system in eukaryotes. In order to protect themselves from RNA virus infection, cells have a complex system for detecting, attacking, and destroying viral RNA. The system takes advantage of a subtle vulnerability of the infecting virus: At some point, in order to multiply within the infected cell, the virus must express its genes—it must make complementary copies of them that can serve as messenger RNAs to direct production of virus proteins.
At that point, while the viral RNA molecule is doublestranded, the virus is vulnerable to attack: At no place in the cell is double-stranded RNA usually found, so by targeting double-stranded RNAs for immediate destruction, a cell can defeat virus infections.
Silencing genes with complementary RNA, dubbed "RNA interference,” offers the exciting hope that successful treatment of many diseases may be literally at our doorstep. First, however, scientists must figure out how to make RNA interference therapies work. They are facing some formidable technical problems, not the least of which is to find a way to deliver the interfering RNA to, and into, the target cells. The problem is that RNA is rapidly broken down in the bloodstream, and most of the body's cells don't readily absorb it, even if it does reach them. Some researchers are attempting to package the RNA into viruses, although as you will learn in chapter 13, gene therapies that have attempted this approach can trigger an immune response and could even cause cancer. Gene therapy researchers have been seeking safer virus gene-delivery vehicles; what they learn will surely be put to good effect.
One interesting alternative approach is to modify the RNA to protect it and make it more easily taken up by cells. This work focuses on the mRNA that encodes apolipoprotein B, a molecule involved in the metabolism of cholesterol. High levels of apolipoprotein are found in people with high levels of cholesterol, associated with increased risk of coronary heart disease. Interfering RNAs that target apolipoprotein B mRNA result in destruction of the mRNA, and lower levels of cholesterol. To effectively deliver it to the body's tissues, researchers simply attached a molecule of cholesterol to each interfering RNA molecule, as shown above. Levels of apolipoprotein B were reduced 50% to 70%, and blood cholesterol levels plummeted downwards, to the same levels seen in cells from which the apolipoprotein B gene had been deleted. It is not clear if this approach will work for many other RNAs, but it looks promising.
A second major problem confronting those seeking to develop successful therapies based on RNA silencing of troublesome genes is one of specificity. It is very important that only the target gene be silenced. Before carrying out clinical trials involving large numbers of people, it is imperative that we be sure the interfering RNA will not shut down vital human genes as well as the targeted virus or cancer genes. Some studies suggest this will not be a problem, while in others a range of "off-target” genes seem to be affected. This possibility will have to be carefully evaluated for each new therapy being developed.