Junk DNA: A Journey Through the Dark Matter of the Genome (16 page)

The number of studies that report associations between long non-coding RNA expression and cancer are growing all the time. Intriguing data are also emerging from genetic studies of cancers. Some cancers are caused by a single really strong mutation which is passed on within a family. Probably the best-known example
is the mutated BRCA1 gene which puts women at very high risk of aggressive breast cancer. It was knowing that she had a mutation in this gene that led the actress Angelina Jolie to elect for a double mastectomy in 2013. Such very strong single gene mutations are pretty rare in cancer. But studies have shown that quite a number of cancers do have a genetic component. The problem has been that when scientists mapped where the genetic variations were that were associated with cancer risk, they were frequently in regions of the genome where there were no protein-coding genes. Of just over 300 genetic variations linked to cancer, only 3.3 per cent changed amino acids in a protein, and over 40 per cent were located in regions between classical protein-coding genes. In these situations the variations may be affecting not protein-coding genes but long non-coding RNAs. Recent studies have confirmed this is the case for some of these variations in at least two cancer types (papillary thyroid cancer and prostate cancer).
¹⁷

Encouragingly, we are also beginning to gather functional data that shows in some cases that these relationships are more than just associations, that the long non-coding RNAs are themselves causing alterations in the behaviour of the cancer cells.

There is a long non-coding RNA whose expression is increased in prostate cancer. This over-expression causes decreased expression of key proteins that normally hold cells back from proliferating too fast.
^18
,
19
Over-expression of this long non-coding RNA is therefore essentially like releasing the handbrake on a car parked facing down a hill. The long non-coding RNA that causes skeletal deformations when it is knocked out in developing mice is over-expressed in a variety of cancers including liver,
²⁰
colorectal,
²¹
pancreatic
²²
and breast
²³
and its over-expression is associated with poor prognosis for the patients. Studies using cancer cells in culture in the lab suggest that the over-expression of this long non-coding RNA may make the cells more likely to migrate and invade other parts of the body.

Some of the strongest data confirming that long non-coding RNAs are actively involved in cancer, rather than just carried along for the ride, come from prostate cancer. When prostate cancer begins to develop, its growth depends on the male hormone, testosterone. Testosterone binds to a receptor and this leads to activation of various genes that promote cell proliferation. Testosterone binding to its receptor is like you putting your foot down on the accelerator pedal of your car. Prostate cancer is initially treated using drugs that stop the hormone binding to its receptor. This is like having something between your foot and the accelerator pedal, so that you can’t press down on it to make the car go faster.

But over time, the cancer cell frequently finds a way around this. The hormone receptor finds ways of activating genes irrespective of whether there is testosterone around or not. It’s as if someone has put a bag of sugar on top of the accelerator. The pedal is always pressed down and speeding up the car, even if you have your feet on the dashboard. Two long non-coding RNAs that are highly over-expressed in aggressive prostate cancer have been shown to play a critical role in this process. They assist the receptor, driving gene expression even when there is no hormone around, and accelerating cell proliferation. They play the role of the bag of sugar in the car simile. If expression of these specific long non-coding RNAs is knocked down in cancer models, the tumours show a really dramatic decrease in growth, supporting the critical role of these molecules.
²⁴

Another long non-coding RNA has also been implicated in prostate cancer. The higher the levels of this long non-coding RNA, the more aggressive the cancer, the shorter the recurrence time after treatment and the greater the risk of death. Knockdown of this long non-coding RNA has a similar protective effect in cancer models to that described above, but in this case the effects do not seem to be due to interactions with the testosterone receptor.
²⁵
This indicates that long non-coding RNAs may affect cancer progression in different ways, even in one tumour type.

Long RNAs and the brain

It isn’t just cancer specialists who are interested in the functions of these molecules. More long non-coding RNAs are expressed in the brain than any other tissue (with the possible exception of the testes).
²⁶
Some have been conserved from birds to humans, with expression patterns that occur in the same regions and at the same developmental stages. These may have conserved functions, perhaps in normal brain development. However, many of the long non-coding RNAs expressed in the brain are specific to humans or primates, and this has led researchers to wonder if they could be responsible, at least in part, for the hugely complex cognitive and behavioural functions found in higher primates.
²⁷

A long non-coding RNA has been identified that influences how the cells in the brain form connections with each other.
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Another long non-coding RNA, which has evolved since we diverged from the other great apes, may be involved in regulating a gene that is required for the unique developmental processes that generate the human cortex.
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The examples above all suggest that long non-coding RNAs play beneficial roles in the brain. But they may also be implicated in pathology as well as in health. Alzheimer’s disease is the devastating dementia which is usually associated with ageing. Because the human population is generally living longer, Alzheimer’s disease is becoming increasingly common. The World Health Organization estimates that over 35 million people worldwide are suffering from dementia, and that this number will double by 2030.
³⁰
There is no cure, and even the drugs that are available, which slow down the clinical progression, don’t stop it altogether, let alone reverse it. The emotional and economic costs of this condition are enormous,
but progress in treating it is horribly slow. This is partly because our understanding of what exactly is going wrong in the brain cells of sufferers is still poor.

We are fairly confident that we know that at least one important step in the process is the production of insoluble plaques in the brain, which can be detected at autopsy. These are made of mis-folded proteins, one of the most important of which is called beta-amyloid. This is generated when an enzyme called BACE1 slices up a larger protein. A long non-coding RNA is produced from the same place in the genes as BACE1, but from the opposite DNA strand, rather like the relationship between Xist and Tsix.

The long non-coding RNA and the standard BACE1 messenger RNA bind to each other. This makes the BACE1 messenger RNA more stable so it stays in the cell for longer. Because it stays around for longer, the cell can generate more copies of the BACE1 protein. This leads to increased production of the beta-amyloid that is essential for the formation of the plaques.
³¹

It’s been reported that the levels of this long non-coding RNA are increased in the brains of patients with Alzheimer’s disease, but it’s difficult to interpret these data. This could just be a consequence of increased expression in that region generally. Remember the earlier analogy – the more you chop up logs, the more sawdust you create. But researchers managed to find a way of specifically decreasing the expression of just the long non-coding RNA in a mouse model which frequently develops Alzheimer’s pathology. The knockdown of the long non-coding RNA resulted in decreased BACE1 protein and fewer beta-amyloid plaques. This supports the idea that the long non-coding RNA may play a causative role in this devastating disease.
³²

It’s not just the central nervous system that can be influenced by long non-coding RNAs. Neuropathic pain is a condition in which the sufferer feels pain, even when there is no physical stimulus. It’s caused by abnormal electrical activity in the nerves that conduct
signals from the periphery of the body into the central nervous system (brain and spinal cord). It can be a very distressing condition for sufferers, and normal painkillers such as aspirin or paracetamol don’t really help. It’s often not clear why the nerves are behaving abnormally. Recent work has suggested that in some cases it may be due to increased levels of a long non-coding RNA changing the expression levels of one of the electrical channels. It does this by binding to the messenger RNA molecule that codes for the channel, altering its stability and hence changing the amount of protein produced.
³³

The types of conditions in which it’s been claimed long non-coding RNAs play a role is growing all the time.
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But the controversy remains over how functional and important these long non-coding RNAs really are. Can they really be as important as proteins? Perhaps on an individual basis the answer is usually ‘no’ unless we are dealing with a molecule as unequivocally vital as Xist. But thinking about the impact in terms of single long non-coding RNAs may be missing the point.

A recent commentary suggested that ‘A distinct possibility is that many of the long transcripts are, at best, nudgers and tweakers of genome management, rather than switches per se.’
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But the greatest complexity and options for flexibility come not from on/off or black/white but from subtle changes in sound levels or from multiple shades of grey. Biologically, we may owe an awful lot to our nudges and tweaks.

Footnotes

^a
This is a famous thought experiment. No actual frogs were harmed in the creation of this anecdote.

^b
They are known as linc RNAs, which stands for long intergenic non-coding RNAs.

9. Adding Colour to the Dark Matter

In biology, the question
What does something do?
is almost always followed by the question
How does it do it?
. We know what long non-coding RNAs are, and we know at least some of what they do – they regulate gene expression. So the perfectly reasonable question is, how do they do that?

There’s not going to be one answer to that question. There are thousands and thousands of long non-coding RNAs produced from the human genome, and they almost certainly won’t all work the same way. But certain themes are starting to emerge.

One of the most important themes relates to a feature we have already encountered, in Chapter 6, on centromeres and their role in cell division. If we look back to Figure 6.3 (
page 70
) we can remind ourselves that the DNA in our cells is wrapped around bundles of eight histone proteins. So far we have referred to these as packaging proteins, but they actually play far more complex roles than that. Our cells can amend the histone proteins, or the DNA itself. They do this by adding small chemical groups to them. These chemical additions don’t change the sequence of a gene. The gene will still code for the same RNA molecule, and the same protein (if it is a protein-coding gene). But the modifications alter the likelihood that a specific gene will be expressed. The modifications are able to do this because they in turn act as binding sites for other proteins. The modifications are the first attachment sites for the build-up of large complexes of proteins that ultimately either switch a gene off or on.

These changes to DNA and its associated proteins are known as epigenetic modifications.
¹
Epi
comes from Greek and means ‘at’, ‘on’, ‘in addition to’, ‘as well as’. These modifications are present in addition to the genetic sequence. The easiest modification to understand is the one that is deposited on DNA itself. By far the most common modification to DNA happens when a C base is followed by a G base. This sequence is called CpG, and enzymes in the cell are able to add a modification here. A chemical group called methyl can be added to the C. Methyl is formed of just one carbon atom and three hydrogen atoms and it’s very small. Sticking one of these on a C base is like sticking a clover leaf on the side of a sunflower bloom.

If there are a lot of CpG motifs in a stretch of DNA, there are lots of sites where the methyl group can be added epigenetically. This attracts proteins that repress expression of that gene. In extreme cases, where there are lots of CpG motifs in close proximity, DNA methylation can have an exceptionally profound effect. Essentially, the DNA changes its shape and the gene is completely switched off. Remarkably, it can be switched off not just in that cell, but in all the daughter cells that are created when it divides. In non-dividing cells, such as the neurons in our brains, these patterns of DNA methylation may be established while we are in the womb. Many of them will still be in place 100 years later, if we make it that far.

The realisation that DNA methylation could switch genes off more or less permanently during the lifetime of an individual caused great excitement. This was because it finally gave scientists a mechanism to probe something that had been puzzling everyone for decades. Essentially we have known for a long time that not everything can be explained by genetics. We know this because there are a lot of situations where two things are genetically identical and yet the two things are different. When a caterpillar pupates and then turns into a butterfly, it continues to use the
same genome. Genetically identical mice, reared under completely standard laboratory conditions, aren’t all the same weight.