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The wander stuff
The wander stuff
U
Renaud Vigourt
The RNA in cells doesn’t always stay put, finds Colin Barras. It can travel far afield, influencing other cells and even other animals
42 | NewScientist | 13 September 2014
NDER the soil of the cornfield, the rootworm larvae emerge from their eggs and crawl in search of roots to munch on. But their mother chose the wrong field to lay her eggs in. There’s something special about the maize here – it’s armed with a smart weapon designed to target the rootworms. As the larvae feed, this weapon is released from the plant and enters their gut cells. There it halts production of a vital protein by blocking one specific gene. In the hours that follow, this “gene silencing” weapon spreads to other cells in the larvae’s bodies. At first there is no noticeable effect – the larvae still have reserves of the protein. But within a few days they start dying. Ten days later they are all gone. This weapon is no ordinary pesticide. It is made of a most surprising substance: RNA. This less-famous relative of DNA has turned out be an extraordinarily versatile molecule. Of the many recent discoveries about it, though, the most surprising is surely that it doesn’t always stay put in cells – it can go walkabout. Some forms of the molecule leave cells and go travelling, carrying vital information that can influence other cells in the same body and, astonishingly, even other organisms. We’ve only just begun studying this phenomenon but it’s already clear that it
will be incredibly useful. Not only can these wandering RNAs be used as pesticides, they can also protect simple animals like honeybees from viruses. The big question, of course, is whether we too can be influenced by foreign RNAs. The first sign that RNA had hidden powers came from a paradoxical result. In the 1990s, a team of biologists studying the colour of flowers assumed, not unreasonably, that giving petunias an extra copy of a gene involved in making a purple pigment would make the flowers darker. Instead, many were entirely white. It gradually became clear that the biologists had inadvertently triggered a previously unknown viral defence mechanism, now called RNA interference. The genomes of many viruses are just a single strand of RNA. When they invade cells, they immediately start making copies of their genome. The first step in copying RNA is to make a complementary strand – that is, to make a double-stranded RNA molecule. It is this that the plant recognises as foreign. The really clever part is that rather than just destroy this RNA, cells hijack it to seek out and destroy any RNAs with the same sequence. This is done by cutting up the viral RNA to create short interfering RNA molecules, called
siRNAs, that bind to matching RNAs, marking them out for destruction by the plant. It’s a bit like cutting up an enemy soldier’s uniform and handing it out to troops along with orders to attack anyone wearing that kind of uniform. The petunias in the original experiment had misidentified the added gene as a dangerous viral molecule and diced it up into siRNA. Because the added gene was a copy of one of the plant’s own genes, the siRNA ended up silencing both copies in its confused attempt to fend off the phantom virus. Consequently, no protein was made and the petunia flowers remained pigment-free. It was soon discovered that RNA interference could be triggered in simple animals, too, and that it has evolved into more than just a defence mechanism: some organisms exploit it to control their own genes within a cell. When it was shown to work in mammals as well, albeit in a more limited way, there was great excitement among biologists. Here was a method that could potentially help treat all kinds of diseases.
“It’s like giving troops enemy uniforms along with orders to kill anyone wearing them”
Meanwhile, as far back as 1997, at the Sainsbury Laboratory in Norwich, UK, David Baulcombe and his then-PhD student Olivier Voinnet noticed that gene silencing had a mysterious ability to spread throughout a plant. This makes sense, though, says Voinnet, now at the Swiss Federal Institute of Technology Zurich, for a form of immunity. “Just think about it as an antiviral response mechanism – the RNA moves ahead of the infection front.”
Organism jump In many animals, too, RNA interference has been found to travel through tissue and influence cells far from the actual site of infection. For researchers who thought they understood RNA, the fact that some of it goes travelling through the body came as a surprise. “These were truly shocking observations at the time,” says Eric Miska, who studies RNA at the University of Cambridge. Given that cells use RNA interference to control their own genes, the ability of RNA to travel around the body suggests it might be used to influence the activity of other cells. And there is an even more intriguing possibility: that it travels between organisms. In 1998, a team led by Andrew Fire at the
Carnegie Institution of Washington in Baltimore, Maryland, and Craig Mello at University of Massachusetts Medical School in Worcester found that simply soaking worms in water carrying double-stranded RNA was enough to trigger RNA interference. Next Fire and his colleague Lisa Timmons showed that feeding the worms bacteria that had been engineered to produce this RNA worked too. Fifteen years on, it is now clear that many invertebrates, from worms to insects, allow their food to manipulate their genes. Many in the field remain baffled by the discovery. “Why would an animal want to do this? It still makes no sense,” says Craig Hunter at Harvard University, who helped discover how the RNA gets absorbed by the worms. “It’s likely not the animal’s intention,” Hunter says. “They are taking up nutrients and other signals from their food – somehow double-stranded RNA gets included in that. It may be because double-stranded RNA is remarkably robust in a way that singlestranded RNA is not.” > 13 September 2014 | NewScientist | 43
The big question is whether some organisms have evolved to exploit this vulnerability. Do some parasites manipulate their hosts by injecting RNAs into them, for instance? We just don’t know, but there are some hints that they do. This year it was reported that a parasitic plant called the strangleweed exchanges large quantities of RNAs with its host plant, for example, but it’s not yet clear why this occurs. Whatever happens in nature, it seems clear that we can exploit RNA interference to trick organisms into attacking their own genes, to kill troublesome weeds or insect pests (New Scientist, 20 March 2010, p 34). One way to do this is to engineer plants to make specific RNAs. Many teams around the world are working on this, but biotech giant Monsanto is leading the field. “We see this as one of the next major breakthroughs in agriculture,” says William Moar, a researcher at the company. Monsanto has long sold corn genetically engineered to produce the insect-killing Bt toxin, but now resistance is emerging. So the company has created a strain that also produces double-stranded RNA targeting a crucial rootworm gene. It will go on sale as SmartStax Pro by the end of the decade. 44 | NewScientist | 13 September 2014
Monsanto’s enthusiasm will make some suspicious, but many biologists see RNAi as far preferable to drenching crops in chemical pesticides that can persist in the environment and end up harming a wide range of animals, not to mention farm workers. “It’s very specific,” says Wayne Hunter at the US Department of Agriculture’s research lab in Fort Pierce, Florida. “If you ever wanted a pesticide that was more focused, more environmentally friendly – this is it.”
Spray on And it doesn’t necessarily have to involve modifying crops genetically. “When I consulted with the citrus and grape companies they specifically wanted a nontransgenic solution so they could market their juice as 100 per cent natural,” says Wayne Hunter. That means spraying crops with fluid containing RNA for a faster, more flexible effect than spending years engineering the plant to produce it.
This used to be out of question, because double-stranded RNA was so expensive to make. “You could spend $600 on a kit and get 2 milligrams of double-stranded RNA – that’s enough to influence about 20 insects,” says Hunter. Things changed in 2009, when a USbased company called Beeologics developed a cheap way to mass-produce it. “That was the year that the world changed for entomology,” says Hunter, who worked with Beeologics before it was acquired by Monsanto in 2011. What’s more, RNAs can be used to “vaccinate” invertebrates against viruses as well as to kill them. Given that RNA interference evolved as a defence mechanism, Wayne Hunter’s team tried creating an RNA to target a virus implicated in colony collapse disorder – a mysterious phenomenon that has led to alarming drops in bee populations. The approach worked: feeding honeybees with a sugar solution containing the RNAs helped the bees fend off infection. “Normally entomologists like me work out ways to kill insects,” says Hunter. “Here we are
Although our cells do not generate siRNA molecules in anything like the numbers that plants, worms and insect cells do, they do generate superficially similar short RNAs called microRNA (miRNA). These molecules help regulate gene expression (New Scientist, 28 June 2008, p 44). And like plant siRNAs, they can move: a number of studies over the past decade have found tiny quantities of these miRNAs in human blood, urine and breast milk.
Hidden messages
“Feeding RNAs to bees helped them fend off infection” saying this technology can be used to protect insect species too.” More recently, the approach has been used to protect other commercially important species against viral infection – including silkworms and farmed Pacific white shrimp. Nevertheless, many researchers are far from convinced that we understand enough about the possible effects of exploiting RNA interference, particularly when used as a pesticide. “To attempt to use this technology at this current stage of understanding would be more naive than our use of DDT in the 1950s,” the US National Honey Bee Advisory Board warned last year. Undoubtedly, there are still many questions to be answered. A big one is whether RNAs really will affect only a single species, or if an RNA intended to kill one insect species could affect others if they are somehow exposed. And because RNAi works, after a fashion, in mammals, and insects are vulnerable to wandering RNA in the food they eat, does that mean it might have an effect on us?
A study published in 2011 suggested that might be the case: fragments of RNA from some vegetables and cereals could be found in human blood, it claimed, and some of those fragments were able to change gene expression in mammal cells growing in a dish. But a spate of subsequent studies suggested otherwise. That is not surprising given that medical researchers are finding it hard to trigger RNA interference in mammals. The long double-stranded RNAs that work a treat in invertebrates don’t work in mammals at all. To make them work, you’ve got to get short siRNAs inside cells, and even injecting RNAs directly into the bloodstream isn’t enough. So it seems that mammals, at least, will not be susceptible to RNA pesticides. “Nonetheless, as a scientist you can’t be too sceptical,” says Voinnet. “You have to be open and admit there is a possibility. This is why my group, among many others, are now pooling efforts as part of a big project to address this and other issues of RNA interference movement in mammals.” Voinnet is referring to a multimillion-dollar project funded by the US National Institutes of Health, which includes Craig Hunter’s group. One of its focuses is a form of wandering RNA that we know for sure is found in our bodies.
But do miRNAs carry signals between cells in the same way that siRNA does in plants or worms? The consensus right now is that they don’t, because the quantities are so small. “Even within a cell, you need high concentrations of miRNA before they can actually do anything,” says Miska. A leading theory is that miRNAs can be found in these bodily fluids simply because the cells they formed inside have grown old, died and spilled their contents. In other words, our wandering miRNA might simply be homeless. There could yet be a twist in the tale, though. Homeless and voiceless it might be, but wandering miRNA might not be useless. It might, like the siRNAs travelling through plant tissue, carry with it a message about the health of the cell in which it formed. And even if that message is too subtle to be heard by the other cells in our body, today’s sophisticated medical tools might finally give it a voice. “If there is any information in the microRNAs floating around our tissue it would offer a great diagnostic tool,” says Miska. Few now doubt that wandering RNA, or exRNA as some have now begun to call this extracellular RNA, has the potential to revolutionise agriculture and insect conservation efforts. Until recently, it had mostly been ignored by medical researchers. “They said it only happens in plants, insects and nematodes,” says Miska. But if exRNA can tell us about, or even influence, our own bodies, it could prove to be a boon to medicine as well. n Colin Barras is a freelance writer based in Michigan, US 13 September 2014 | NewScientist | 45
U
Renaud Vigourt
The RNA in cells doesn’t always stay put, finds Colin Barras. It can travel far afield, influencing other cells and even other animals
42 | NewScientist | 13 September 2014
NDER the soil of the cornfield, the rootworm larvae emerge from their eggs and crawl in search of roots to munch on. But their mother chose the wrong field to lay her eggs in. There’s something special about the maize here – it’s armed with a smart weapon designed to target the rootworms. As the larvae feed, this weapon is released from the plant and enters their gut cells. There it halts production of a vital protein by blocking one specific gene. In the hours that follow, this “gene silencing” weapon spreads to other cells in the larvae’s bodies. At first there is no noticeable effect – the larvae still have reserves of the protein. But within a few days they start dying. Ten days later they are all gone. This weapon is no ordinary pesticide. It is made of a most surprising substance: RNA. This less-famous relative of DNA has turned out be an extraordinarily versatile molecule. Of the many recent discoveries about it, though, the most surprising is surely that it doesn’t always stay put in cells – it can go walkabout. Some forms of the molecule leave cells and go travelling, carrying vital information that can influence other cells in the same body and, astonishingly, even other organisms. We’ve only just begun studying this phenomenon but it’s already clear that it
will be incredibly useful. Not only can these wandering RNAs be used as pesticides, they can also protect simple animals like honeybees from viruses. The big question, of course, is whether we too can be influenced by foreign RNAs. The first sign that RNA had hidden powers came from a paradoxical result. In the 1990s, a team of biologists studying the colour of flowers assumed, not unreasonably, that giving petunias an extra copy of a gene involved in making a purple pigment would make the flowers darker. Instead, many were entirely white. It gradually became clear that the biologists had inadvertently triggered a previously unknown viral defence mechanism, now called RNA interference. The genomes of many viruses are just a single strand of RNA. When they invade cells, they immediately start making copies of their genome. The first step in copying RNA is to make a complementary strand – that is, to make a double-stranded RNA molecule. It is this that the plant recognises as foreign. The really clever part is that rather than just destroy this RNA, cells hijack it to seek out and destroy any RNAs with the same sequence. This is done by cutting up the viral RNA to create short interfering RNA molecules, called
siRNAs, that bind to matching RNAs, marking them out for destruction by the plant. It’s a bit like cutting up an enemy soldier’s uniform and handing it out to troops along with orders to attack anyone wearing that kind of uniform. The petunias in the original experiment had misidentified the added gene as a dangerous viral molecule and diced it up into siRNA. Because the added gene was a copy of one of the plant’s own genes, the siRNA ended up silencing both copies in its confused attempt to fend off the phantom virus. Consequently, no protein was made and the petunia flowers remained pigment-free. It was soon discovered that RNA interference could be triggered in simple animals, too, and that it has evolved into more than just a defence mechanism: some organisms exploit it to control their own genes within a cell. When it was shown to work in mammals as well, albeit in a more limited way, there was great excitement among biologists. Here was a method that could potentially help treat all kinds of diseases.
“It’s like giving troops enemy uniforms along with orders to kill anyone wearing them”
Meanwhile, as far back as 1997, at the Sainsbury Laboratory in Norwich, UK, David Baulcombe and his then-PhD student Olivier Voinnet noticed that gene silencing had a mysterious ability to spread throughout a plant. This makes sense, though, says Voinnet, now at the Swiss Federal Institute of Technology Zurich, for a form of immunity. “Just think about it as an antiviral response mechanism – the RNA moves ahead of the infection front.”
Organism jump In many animals, too, RNA interference has been found to travel through tissue and influence cells far from the actual site of infection. For researchers who thought they understood RNA, the fact that some of it goes travelling through the body came as a surprise. “These were truly shocking observations at the time,” says Eric Miska, who studies RNA at the University of Cambridge. Given that cells use RNA interference to control their own genes, the ability of RNA to travel around the body suggests it might be used to influence the activity of other cells. And there is an even more intriguing possibility: that it travels between organisms. In 1998, a team led by Andrew Fire at the
Carnegie Institution of Washington in Baltimore, Maryland, and Craig Mello at University of Massachusetts Medical School in Worcester found that simply soaking worms in water carrying double-stranded RNA was enough to trigger RNA interference. Next Fire and his colleague Lisa Timmons showed that feeding the worms bacteria that had been engineered to produce this RNA worked too. Fifteen years on, it is now clear that many invertebrates, from worms to insects, allow their food to manipulate their genes. Many in the field remain baffled by the discovery. “Why would an animal want to do this? It still makes no sense,” says Craig Hunter at Harvard University, who helped discover how the RNA gets absorbed by the worms. “It’s likely not the animal’s intention,” Hunter says. “They are taking up nutrients and other signals from their food – somehow double-stranded RNA gets included in that. It may be because double-stranded RNA is remarkably robust in a way that singlestranded RNA is not.” > 13 September 2014 | NewScientist | 43
The big question is whether some organisms have evolved to exploit this vulnerability. Do some parasites manipulate their hosts by injecting RNAs into them, for instance? We just don’t know, but there are some hints that they do. This year it was reported that a parasitic plant called the strangleweed exchanges large quantities of RNAs with its host plant, for example, but it’s not yet clear why this occurs. Whatever happens in nature, it seems clear that we can exploit RNA interference to trick organisms into attacking their own genes, to kill troublesome weeds or insect pests (New Scientist, 20 March 2010, p 34). One way to do this is to engineer plants to make specific RNAs. Many teams around the world are working on this, but biotech giant Monsanto is leading the field. “We see this as one of the next major breakthroughs in agriculture,” says William Moar, a researcher at the company. Monsanto has long sold corn genetically engineered to produce the insect-killing Bt toxin, but now resistance is emerging. So the company has created a strain that also produces double-stranded RNA targeting a crucial rootworm gene. It will go on sale as SmartStax Pro by the end of the decade. 44 | NewScientist | 13 September 2014
Monsanto’s enthusiasm will make some suspicious, but many biologists see RNAi as far preferable to drenching crops in chemical pesticides that can persist in the environment and end up harming a wide range of animals, not to mention farm workers. “It’s very specific,” says Wayne Hunter at the US Department of Agriculture’s research lab in Fort Pierce, Florida. “If you ever wanted a pesticide that was more focused, more environmentally friendly – this is it.”
Spray on And it doesn’t necessarily have to involve modifying crops genetically. “When I consulted with the citrus and grape companies they specifically wanted a nontransgenic solution so they could market their juice as 100 per cent natural,” says Wayne Hunter. That means spraying crops with fluid containing RNA for a faster, more flexible effect than spending years engineering the plant to produce it.
This used to be out of question, because double-stranded RNA was so expensive to make. “You could spend $600 on a kit and get 2 milligrams of double-stranded RNA – that’s enough to influence about 20 insects,” says Hunter. Things changed in 2009, when a USbased company called Beeologics developed a cheap way to mass-produce it. “That was the year that the world changed for entomology,” says Hunter, who worked with Beeologics before it was acquired by Monsanto in 2011. What’s more, RNAs can be used to “vaccinate” invertebrates against viruses as well as to kill them. Given that RNA interference evolved as a defence mechanism, Wayne Hunter’s team tried creating an RNA to target a virus implicated in colony collapse disorder – a mysterious phenomenon that has led to alarming drops in bee populations. The approach worked: feeding honeybees with a sugar solution containing the RNAs helped the bees fend off infection. “Normally entomologists like me work out ways to kill insects,” says Hunter. “Here we are
Although our cells do not generate siRNA molecules in anything like the numbers that plants, worms and insect cells do, they do generate superficially similar short RNAs called microRNA (miRNA). These molecules help regulate gene expression (New Scientist, 28 June 2008, p 44). And like plant siRNAs, they can move: a number of studies over the past decade have found tiny quantities of these miRNAs in human blood, urine and breast milk.
Hidden messages
“Feeding RNAs to bees helped them fend off infection” saying this technology can be used to protect insect species too.” More recently, the approach has been used to protect other commercially important species against viral infection – including silkworms and farmed Pacific white shrimp. Nevertheless, many researchers are far from convinced that we understand enough about the possible effects of exploiting RNA interference, particularly when used as a pesticide. “To attempt to use this technology at this current stage of understanding would be more naive than our use of DDT in the 1950s,” the US National Honey Bee Advisory Board warned last year. Undoubtedly, there are still many questions to be answered. A big one is whether RNAs really will affect only a single species, or if an RNA intended to kill one insect species could affect others if they are somehow exposed. And because RNAi works, after a fashion, in mammals, and insects are vulnerable to wandering RNA in the food they eat, does that mean it might have an effect on us?
A study published in 2011 suggested that might be the case: fragments of RNA from some vegetables and cereals could be found in human blood, it claimed, and some of those fragments were able to change gene expression in mammal cells growing in a dish. But a spate of subsequent studies suggested otherwise. That is not surprising given that medical researchers are finding it hard to trigger RNA interference in mammals. The long double-stranded RNAs that work a treat in invertebrates don’t work in mammals at all. To make them work, you’ve got to get short siRNAs inside cells, and even injecting RNAs directly into the bloodstream isn’t enough. So it seems that mammals, at least, will not be susceptible to RNA pesticides. “Nonetheless, as a scientist you can’t be too sceptical,” says Voinnet. “You have to be open and admit there is a possibility. This is why my group, among many others, are now pooling efforts as part of a big project to address this and other issues of RNA interference movement in mammals.” Voinnet is referring to a multimillion-dollar project funded by the US National Institutes of Health, which includes Craig Hunter’s group. One of its focuses is a form of wandering RNA that we know for sure is found in our bodies.
But do miRNAs carry signals between cells in the same way that siRNA does in plants or worms? The consensus right now is that they don’t, because the quantities are so small. “Even within a cell, you need high concentrations of miRNA before they can actually do anything,” says Miska. A leading theory is that miRNAs can be found in these bodily fluids simply because the cells they formed inside have grown old, died and spilled their contents. In other words, our wandering miRNA might simply be homeless. There could yet be a twist in the tale, though. Homeless and voiceless it might be, but wandering miRNA might not be useless. It might, like the siRNAs travelling through plant tissue, carry with it a message about the health of the cell in which it formed. And even if that message is too subtle to be heard by the other cells in our body, today’s sophisticated medical tools might finally give it a voice. “If there is any information in the microRNAs floating around our tissue it would offer a great diagnostic tool,” says Miska. Few now doubt that wandering RNA, or exRNA as some have now begun to call this extracellular RNA, has the potential to revolutionise agriculture and insect conservation efforts. Until recently, it had mostly been ignored by medical researchers. “They said it only happens in plants, insects and nematodes,” says Miska. But if exRNA can tell us about, or even influence, our own bodies, it could prove to be a boon to medicine as well. n Colin Barras is a freelance writer based in Michigan, US 13 September 2014 | NewScientist | 45