The amino acid alphabet

Amino acid alphabet soup

Via Astrobiology Magazine, 8/19/11

All living creatures on this planet use the same 20 amino acids, even though there are hundreds available in nature. Scientists therefore have wondered if life could have arisen based on a different set of amino acids. And what's more, could life exist elsewhere that utilizes an alternate collection of building blocks?

It really is rather remarkable that such a small subset of possible amino acids make up (almost) all the proteins in every known living organism on the planet. What enforces this strict discipline is the fact that all life forms on Earth use the same genetic code – a remarkable fact in itself – and this code does not specify any amino acids other than the same 20 ones. The way the code works makes substitutions impossible.

The reason for this inflexibility lies in the nature of transfer RNA, which is a critical part of the process in which genetic information encoded in DNA is converted to specific sequences of amino acids making up proteins. The DNA sequence of genes is first transcribed (in a process that is actually rather complicated) into another form of RNA – messenger RNA. All forms of RNA consist of a sequence of nucleotides, with every 3 nucleotides grouped together into "words". Since there are 4 possible nucleotides, there are 64 (=4³) possible distinct words.

In a molecule of transfer RNA, which typically comprises 73 to 93 nucleotides altogether, the three nucleotides at one end will match the sequence of one particular word of messenger RNA. The other end of the transfer RNA can bind to covalently to only one of 20 possible amino acids, completely ignoring any other amino acids. For any particular one of the 20 amino acids there are usually several different transfer RNAs that the amino acid can bind to, with each type corresponding to a specific 3-letter sequence of nucleotides. In this way there is a established a many-to-1 relationship between the 64 3-letter nucleotide words and the 20 amino acids. This is the genetic code.

The 20 amino acids can be considered as letters of another alphabet, in which sequences of letters (sometimes thousands of each) make up specific proteins. There are several interesting questions about this genetic code. Why are only 20 amino acids used, even though hundreds exist in nature? How did this small subset happen to be chosen – and be the same subset in all living organisms on Earth? If there is life on other planets that still encodes genetic information with DNA and RNA for making proteins, must the same 20 amino acids be used?

There is a range of possible answers to these questions. At one extreme, the subset of amino acids could have come about completely at random, perhaps being the first viable subset that emerged by chance and them became "frozen" in all successor life forms. At the other extreme, it could be that the amino acids actually used are the only ones that are able to build a suitable set of proteins. The intermediate case is that very early in the history of life many different subsets were in use, but in a process of evolution over time, the subset now used proved to be sufficiently superior to all others that it is the only one that survived in the conditions of the time.

Stephen Freeland and Gayle Philip performed a computer study to investigate whether the exact subset of 20 amino acids in the alphabet were more likely to be a completely random selection, or instead to represent a set that emerged as somehow the best suited for constituting the proteins of life on Earth. They reasoned that there were various properties any amino acid could have that would affect its suitability as a constituent of proteins. Among the properties were size and electric charge of the molecule, and the molecule's degree of attraction to water (hydrophilicity).

What they found was that the 20 amino acids actually occurring in proteins had a wide range of values for each of the properties, and that the range of properties was more evenly distributed over the subset than should occur if selection were random. In other words, the building blocks of proteins appear to be especially diverse in order to accommodate a large diversity of proteins that could be useful in living organisms. Thus evolution in the earliest stages of life on Earth probably favored the availability of many types of building blocks.

Abstract: Did evolution select a nonrandom "alphabet" of amino acids?

The last universal common ancestor of contemporary biology (LUCA) used a precise set of 20 amino acids as a standard alphabet with which to build genetically encoded protein polymers. Considerable evidence indicates that some of these amino acids were present through nonbiological syntheses prior to the origin of life, while the rest evolved as inventions of early metabolism. However, the same evidence indicates that many alternatives were also available, which highlights the question: what factors led biological evolution on our planet to define its standard alphabet? One possibility is that natural selection favored a set of amino acids that exhibits clear, nonrandom properties-a set of especially useful building blocks. However, previous analysis that tested whether the standard alphabet comprises amino acids with unusually high variance in size, charge, and hydrophobicity (properties that govern what protein structures and functions can be constructed) failed to clearly distinguish evolution's choice from a sample of randomly chosen alternatives. Here, we demonstrate unambiguous support for a refined hypothesis: that an optimal set of amino acids would spread evenly across a broad range of values for each fundamental property. Specifically, we show that the standard set of 20 amino acids represents the possible spectra of size, charge, and hydrophobicity more broadly and more evenly than can be explained by chance alone.

DNA repair and cancer II

I just wrote about this at some length, here.

It's funny, sometimes, how there are a bunch of results released in a short time period on the same topic. Usually it's because there's a big meeting that covers the topic, or else the editor of some journal wants several papers on the topic.

I've just come across three more papers on the topic and can't recall such a flurry of activity related to it. The papers were all published in different journals, and there doesn't appear to have just been a meeting on the topic, but it's understandably an active area of research.

The issue is whether or not variants of genes for DNA repair proteins make good biomarkers for cancer risk. As I discussed before, the recent meta-analysis suggests that in general this isn't as great a place to look for biomarkers as one might expect.

Compared to the most common allele of a DNA repair gene, less common alleles may correlate with either increased or decreased cancer risk, depending on the type of cancer and type of DNA repair involved.

In neither case can one automatically conclude that the common allele yields protection against cancer. By repairing DNA, the repair enzyme might actually help cancerous cells survive, offsetting any benefit from preventing further genome damage.

As a result, if one finds that a specific allele reduces cancer risk, it could be because the allele is actually less effective at repairing DNA. Conversely, if another allele increases cancer risk, it could be because the allele is too effective at repairing DNA.

Just looking at the situation from an anticancer perspective, when DNA damage is detected, the smart thing to do is to sacrifice the cell via apoptosis. But that's not the way nature looks at things. The type of damage involved may be so common that it's smarter to try to fix it, and then hope for the best cancer-wise. After all, most cancer depends on the presence of other, unrelated and probably very uncommon, kinds of genomic damage.

Research studies in this area really need to try to suss out what is actually happening, and that could be rather difficult. Basically, this is an evolutionary problem, with the outcome depending on what happens, statistically, in millions or billions of cells over a period of time. Which alleles will ultimately win out? The ones trying to fix DNA damage, or the others trying to exploit the damage? All the "players" in this game have different interests at stake.

Progress here may require some heavy-duty computer simulations trying to sort it all out. Cancer research could turn out to be a lot like climate modeling.

What would better understanding mean therapeutically? It could be possible to discover biomarkers – alleles that indicate significant cancer risk. Where serious risk is indicated to exist, then standard interventions like surgery or chemotherapy may be appropriate.

On the other hand, developing drugs that attempt to silence an allele which is found to predict significant cancer risk may not be very rewarding, for all the usual reasons that drugs may fail (e. g. side effects). That's what clinical trials are for – and trials are very expensive.

The first study we'll look at here involves fairly unique circumstances. It does not address many of the issues we'd like to know about, in particular concerning a direct mechanistic relationship between DNA repair and cancer.

But let's look at what it does say, with a view towards where further research could go. The study, first, determines that a specific type of DNA repair ("nucleotide excision repair") rises and falls substantially with the circadian clock. Second, it finds that levels of a specific repair-related protein (xeroderma pigmentosum A, or XPA) also rises and falls with the clock. Third, it shows levels of XPA are directly related to DNA repair activity.

Now, it's well known that certain types of chemotherapy have a circadian dependency. Quoting from the paper, there exists "empirical observation that circadian time of delivery of chemotherapeutic drugs such as cisplatin, whose major DNA lesions are cisplatin-d(GpG) and cisplatin-d(GpXpG) diadducts (6, 7), may be a significant contributing factor to the efficacy of the drug and the severity of its side effects (4, 5)." Thus it's at least plausible that circadian variability of the levels of repair-related proteins could account for this.

However, that hypothesis remains to be checked directly. It will be interesting to see how this develops.

Here's a press release:

Chemotherapy Most Effective At Time Of Day When Particular Enzyme At Lowest Level (1/13/09)

For years, research has hinted that the time of day that cancer patients receive chemotherapy can impact their chances of survival. But the lack of a clear scientific explanation for this finding has kept clinicians from considering timing as a factor in treatment.

Now, a new study from the University of North Carolina at Chapel Hill has suggested that treatment is most effective at certain times of day because that is when a particular enzyme system – one that can reverse the actions of chemotherapeutic drugs – is at its lowest levels in the body. ...

The study, published in the Proceedings of the National Academy of Sciences, provides the first solid evidence that the daily oscillations of the cell's repair machinery can affect the potency of cancer drugs.

Meta-observation: It's apparently difficult to pin down specific mechanisms at work here, so a fair amount of indirect inference is needed to draw conclusions. We'll see the same thing in two other recent studies (below).

Research paper:

Circadian oscillation of nucleotide excision repair in mammalian brain

T.-H. Kang, J. T. Reardon, M. Kemp, A. Sancar (2009). Circadian oscillation of nucleotide excision repair in mammalian brain Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0812638106

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Next up is a a case-control study that looked at nine single-nucleotide polymorphisms (SNPs) of seven DNA repair genes. The researchers were looking for correlations with the occurrence of pancreatic cancer. They studied 734 pancreatic cancer patients and 780 individuals without cancer. The strongest association was with a variant allele of one gene (LIG3), where individuals with the allele were 77% less likely than carriers of the normal gene to have cancer. The next strongest association was with a variant allele of another gene (ATM), carriers of which were in excess of 100% more likely to have cancer. (ATM also figures prominently in the third study, discussed below.)

Further investigation considered other cancer risk factors, such as smoking, heavy alcohol consumption, excess body weight, or diabetes – factors which might be responsible for excess DNA damage. The most significant factor turned out to be diabetes. In individuals with diabetes, carriers of an ATM allele were more than 200% likelier to have cancer, while carriers of a LIG4 allele were more than 100% likelier to have cancer.

So here's a study that successfully found several biomarkers, but involving only a few of the DNA genes studied.

Press release:

Abnormal DNA Repair Genes May Predict Pancreatic Cancer Risk (1/15/09)

Abnormalities in genes that repair mistakes in DNA replication may help identify people who are at high risk of developing pancreatic cancer, a research team from The University of Texas M. D. Anderson Cancer Center reports in the Jan. 15 issue of Clinical Cancer Research.

Defects in these critical DNA repair genes may act alone or in combination with traditional risk factors known to increase an individual's likelihood of being diagnosed with this very aggressive type of cancer. ...

With this in mind, [lead author Donghui] Li and her colleagues set out to identify DNA repair genes that could act as susceptibility markers to predict pancreatic cancer risk. In a case-control study of 734 patients with pancreatic cancer and 780 healthy individuals, they examined nine variants of seven DNA repair genes. The repair genes under investigation were: LIG3, LIG4, OGG1, ATM, POLB, RAD54L and RECQL.

The researchers looked for direct effects of the gene variants (also called single nucleotide polymorphisms) on pancreatic cancer risk as well as potential interactions between the gene variants and known risk factors for the disease, including family history of cancer, diabetes, heavy smoking, heavy alcohol consumption and being overweight.

Research abstract:

DNA Repair Gene Polymorphisms and Risk of Pancreatic Cancer

These observations suggest that genetic variations in DNA repair may act alone or in concert with other risk factors on modifying a patient's risk for pancreatic cancer.

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The last study we'll consider here is a little more tangential to the issue of DNA repair genes and cancer. It's primarily about how defects in DNA repair genes may be responsible for two related neurological diseases – ataxia telangiectasia-like disease (ATLD) and Nijmegen breakage syndrome (NBS).

The two diseases result from defects in the proteins Mre11 and Nbs1 (respectively), which are part of a protein complex called MRN (Mre11-Rad50-Nbs1). MRN is a part of a mechanism that repairs double-stranded DNA breaks.

The research used mouse models. Mice engineered to have an ATLD-like disease had defective genes for Mre11, while those with the NBS-like disease had defective genes for Nbs1. In both cases, DNA-damage stress was induced either by radiation or by knocking out another DNA repair enzyme. The idea was to trigger disease effects due to inadequate damage repair by MRN.

A further relevant fact is that part of the MRN damage-repair process invokes a protein kinase called ATM. Part of the role of ATM is to trigger apoptosis if the damaged DNA cannot be repaired.

For our purposes here, the relevant finding was that neurons of the ATLD mice were resistant to apoptosis, and consequently DNA-damaged neurons survived longer than they should, in view of their defects. The disease pathology results from the persistence of damaged neurons that cannot perform as required. However, neurons of the NBS mice were not unusually resistant to apoptosis, so the neurons died more rapidly, and pathology results from the excessive cell death.

How Defective DNA Repair Triggers Two Neurological Diseases (1/14/09)

To explore the differences between ATLD and NBS, the researchers used mice engineered to have defects in the causative genes, which produce two proteins that help form a critical component of the DNA repair machinery, called the MRN complex. The MRN complex zeroes in on broken DNA segments and attaches to them. It then recruits another important DNA repair protein, called ATM, to launch the repair process. However, if the damage is too severe, ATM may also trigger programmed cell death called apoptosis.

"It happens that defects in ATM also lead to a disease similar to ATLD, highlighting the connections between diseases resulting from defects in this DNA repair pathway," [senior author Peter] McKinnon said.

The mice engineered to mimic ATLD, like their human counterparts, had defective genes that produce a protein called Mre11; while NBS mice were engineered to have defects in the gene for the protein called Nbs1.

The key point may be this: when Mre11 is defective and ATM is then activated, it does not always trigger apoptosis when it should. But when Nbs1 is defective, ATM is able to do its job properly. In any event, when either Mre11 or Nbs1 is defective, MRN does not repair DNA damage as well as it should (in cells other than neurons). This may raise the risk of cancer due to randomly damaged DNA.

"There is a suspicion that people who carry these mutations may be predisposed to cancer and also more susceptible to chemotherapy agents or even to standard X-rays," McKinnon said. "Those agents induce the type of DNA damage that requires the MRN complex and ATM for repair. More generally, studies of the MRN complex and ATM are fundamental to understanding how to prevent changes to DNA that lead to cancer.

"Understanding more about how these proteins signal and interact, and how different cells in the body transduce the DNA damage signal, is of fundamental biological importance," McKinnon said. "This knowledge is necessary not only for understanding DNA repair diseases but for understanding the broader implications of maintaining of the stability of DNA."

Tags: cancer, DNA damage, DNA repair

DNA repair genes and cancer

DNA encodes genes needed for the production of essential proteins in a cell, and this DNA is vulnerable to damage in a number of ways. Perhaps the most common way is a consequence of inevitable errors that occur in the copying of all of a cell's DNA during cell division.

But there are a number of other ways that DNA damage can occur, such as damage due to reactive oxygen species or carcinogenic chemicals, ionizing radiation, or retroviruses, which insert their own DNA at random locations in the genome. (Technically, there's a distinction between DNA damage and DNA mutations, but we'll gloss over that for a moment.)

Most DNA damage affects only a single cell, and then, at most, only in the cell's ability to make certain proteins needed for normal cell function. Damage that occurs in DNA regions that don't affect protein production may have no effect at all. If important proteins are afffected, the cell may be unable to do its job properly, such as being a neuron or muscle cell. At worst, the cell dies, but its function is probably duplicated by billions of other cells in the same tissue or organ. Over a long enough period of time, of course, eventually a large percentage of cells will die due to DNA damage – and then the organism dies of "old age", if nothing more traumatic occurs first.

Against the relative improbability of one DNA damage event having serious consequences for a particular cell has to be weighed the frequency with which damage occurs. Most cells divide fewer than 100 times in their whole lifespan, though many errors can occur during each division. External factors like radiation, however, can cause millions of DNA damage events per day in a single cell. (Don't go sitting in front of an X-ray source all day! Or even under a source of UV radiation, like the Sun.) Internal factors, such as reactive oxygen species produced during normal metabolism, can cause many 1000s of mutations per cell per day, so DNA damage isn't something that cells can just ignore.

There are a couple of exceptions, when DNA damage can be an even more serious problem. One is in case the cell is an egg or sperm cell (a gamete), in a multicellular, sexually-reproducing organism, and the cell goes on to generate a new individual, which will inherit the damaged DNA. Since gametes are only a small proportion of all cells, and very few gametes are ever "lucky" enough to become new individuals, this is a fairly rare event. But it is how errors accumulate in genomes, over long periods of time. Such errors, being random events, are seldom beneficial to a species. But rarely they can be helpful and drive species evolution to adapt to a changing environment.

The other exception, which is much more consequential for the individual in which it occurs, is when the DNA damage affects one of the relatively small number of proteins responsible for keeping cell division and proliferation under control. Then you have a circumstance that can lead to cancer, even if only a single cell is affected to begin with. Clearly, it is in the best interest of the individual, and perhaps the whole species, for cells to have good mechanisms for dealing with the problems caused by DNA damage.

Apart from dealing with the original causes of DNA damage, cells have basically two ways of coping with damage after it occurs.

One sort of mechanism involves being able to detect the presence of DNA damage, and to initiate measures that limit or stop the cell's ability to proliferate, or even cause the cell to die. P53 is perhaps the best known protein involved in this type of mechanism. Compromised p53 production is found in more than half of all cancers. If a gene, in the same cell, that codes for a protein needed to implement one of these mechanisms has been damaged previously, the probability of cancerous proliferation will rise. Mechanisms of this kind are complex. There's a lot that can go wrong, and that's one reason cancer is as common as it is.

The other sort of mechanism is actual DNA repair. It's not so draconian. Such mechanisms attempt to actually correct the DNA damage, returning the DNA to its state before the damage occured. That's great. If it actually works, the cell can survive unharmed, and not pose any extra risk of becoming cancerous.

There are a variety of kinds of DNA damage, and hence a variety of repair mechanisms are needed. Individual bases attached to the nucleic acid backbone can be modified or deleted. The base sequence can be altered by faulty copying. The backbone itself can be broken or warped, preventing expression of some genes. Entire chromosomes, which carry DNA in a compact, packaged form, can be broken or improperly duplicated.

It's easier to recognize that damage has occurred than it is to repair it. Here the distinction between DNA mutations and other kinds of damage becomes relevant. A mutation exists when a base pair on the two strands of DNA is replaced with a different but otherwise normal base pair (i. e. adenine-thymine or guanine-cytosine). The resulting DNA is still technically undamaged, although the protein resulting from a gene with a mutation may not work as well as the unmodified version. In such a case, it is difficult or impossible for the problem to be recognized by DNA repair mechanisms. And it the problem can't be recognized, it certainly can't be repaired.

However, with most DNA damage, one or both strands of the DNA molecule is/are either distorted or broken. In that case, the DNA cannot be properly transcribed into RNA, or the DNA cannot be copied during cell division (or both). But for the same reason that these copying problems occur, it is also possible for appropriate enzymes to recognize that things are not right, and to activate a suitable repair mechanism (even if the repair cannot always succeed).

If, on the other hand, the damage is not repairable, other pathways can be activated to cause cell senescence or apoptosis. This more drastic situation may lead to cancer if there is already a problem with the senescence or apoptosis pathways. However, the research we're about to mention doesn't deal with this case. It's about problems in the repair mechanisms, due to DNA damage or mutations affecting those mechanisms, that lead to repair failure and that, consequently, lead to cancerous cell behavior.

The nature of repairable damage is varied, and the details of repair are quite technical, so we won't go into that here. Suffice it to say that there are effective damage repair mechanisms. What's not clear is whether malfunctions in those mechanisms (resulting from previous uncorrected errors or mutations) frequently result in cancer.

Researchers investigated genes that code for proteins involved in DNA repair. After statistical analysis of many relevant studies they found only two with a significant correlation to cancer:

Few DNA Repair Genes Maintain Association With Cancer In Field Synopsis (12/31/08)

Variants of numerous DNA repair genes initially appeared to be statistically significantly associated with cancer risk in epidemiological studies. When the data from individual studies are pooled, however, few DNA repair gene variants appear truly associated with increased cancer risk, according to a new field synopsis. ...

In the current study, John P. Ioannidis, M.D., of the University of Ioannina School of Medicine in Greece, and colleagues identified 241 previously reported associations between gene variants and the risk of cancer. The team pooled the data from 1,087 data sets and reexamined these associations.

Initially 31 of the 241 associations appeared to be statistically significantly associated with cancer risk in the meta-analysis. However, only two remained statistically significant after the researchers adjusted for multiple comparisons. An XRCC1 allele (-77 T>C) and an allele of ERCC2 (codon 751) were associated with lung cancer risk.

The conclusion of the meta-analysis is that either there's a problem with the way in which candidate genes were selected, or else problems in DNA repair mechanisms do not by themselves play a big role in carcinogenesis.

"The lack of many signals with strong credibility that emerged from our analysis, despite an enormous amount of work in this area over the years, needs careful consideration," the authors write. "The ability of the candidate gene approach to identify genetic risk factors may have been overestimated. Alternatively, the importance of the DNA repair pathway may have been exaggerated. However, there is increasing recognition that genetic risks of cancer conferred by single variants are almost always very modest. This means that even if the DNA repair pathway is essential for carcinogenesis, extremely large-scale evidence would be necessary to establish with high confidence the presence of specific associations."

Even though it seems that genomic studies have failed to turn up important oncogenes among genes that are involved in DNA repair, there is practical significance in these research findings.

For one thing, we should not expect to find useful biomarkers of cancer risk among alleles of DNA repair genes. Likewise, it's probably not worth exploring gene therapy approaches to compensating for malfunctioning DNA repair genes. Instead, what's more likely to succeed is a focus on alleles of genes involved in detection of DNA damage or of genes involved in senescence or apoptosis pathways.

Still, DNA repair genes remain very important. In many types of current cancer therapy, such as chemotherapy or radiotherapy, the intent is explicitly to cause DNA damage in order to bring about senescence or apoptosis. A problem with DNA repair in cancer cells cuts both ways. On one hand, faulty DNA repair leaves more errors uncorrected, exposing the cells to senescence or apoptosis as long as those pathways remain intact. That's good. But on the other hand, it can allow new errors to accumulate, making the cells more vulnerable to compromise of the senescence and apoptosis pathways, hence more likely to proliferate. That's bad.

Perhaps this ambivalence of DNA repair in the cancer process explains the apparent lack of strong correlation between faulty DNA repair and cancer.

Here's another way to think about the "right" way for an organism to defend itself from cancer. Cancer is a problem that's of concern only to multicellular organisms – communities of cells. While the integrity of each cell is certainly important, it's even more important to protect the whole community. A suitable analogy might be that it's more important to the community to have a good fire department as the last line of defense, than to rely on effective sprinkler systems to put out small fires in every separate location. If the fire department itself is compromised, and unable to combat a spreading conflagration, the community as a whole is in serious danger.

Here's the research abstract:

A Field Synopsis on Low-Penetrance Variants in DNA Repair Genes and Cancer Susceptibility

We have conducted meta-analyses of 241 associations between variants in DNA repair genes and cancer and have found sparse association signals with strong epidemiological credibility. This synopsis offers a model to survey the current status and gaps in evidence in the field of DNA repair genes and cancer susceptibility, may indicate potential pleiotropic activity of genes and gene pathways, and may offer mechanistic insights in carcinogenesis.

Update, 1/25/09: There's a significant follow-up on all this here.

Tags: cancer, DNA damage, DNA repair

Non-coding RNA and gene expression

Human DNA consists of about 3.4 billion base pairs. A portion of that is actually genes that code for proteins required by human cells – roughly 20,500 genes. (See here.)

However, it's been recognized for a long time that only about 1.5% of human DNA (in terms of base pairs) actually codes for proteins. Little is known about the purpose (if any) of the remaining 98.5%, even though, by some estimates, 80% of human DNA is transcribed into RNA at some time.

This remainder is often called "junk DNA". But it's also known that a lot of it can't really be "junk", and must serve some useful purpose, because the sequences of large portions of it are highly conserved in evolution, being found almost unchanged in the genomes of human ancestors going back hundreds of millions of years.

Some of the 98.5% really does seem to be without useful function, consisting of stuff like transposons, which are DNA sequences that seem to be copied repeatedly and randomly into various parts of the genome (over evolutionary time spans)

The function of other portions of that 98.5% includes such things as introns found within genes, gene regulatory sequences, and "RNA genes" that code for various kinds of RNA that doesn't wind up being translated into proteins.

Such non-coding RNA can be further classified into things like ribosomal RNA, microRNA, small interfering RNA, and "long non-coding RNA".

This last, known as "long ncRNA" for short, is especially intriguing, because some studies have shown that there may be roughly four times as much of it (in base units) as there is of messenger RNA that is ultimately translated into proteins.

Even though a lot of these long ncRNAs are routinely found floating around inside cells, we're still in the dark about what, if anything, they actually do. But some recent research has revealed a little more about some long ncRNAs:

Early-stage Gene Transcription Creates Access To DNA (10/6/08)

Previously thought to be inert carriers of the genetic instructions from DNA, so-called non-coding RNAs turn out to reveal a novel mechanism for creating access to DNA required by transcriptional activation proteins for successful gene expression, according to Boston College Biology Professor Charles Hoffman, a co-author of the study with researchers from two Japanese universities. ...

Hoffman and his colleagues examined how the yeast cell senses its cellular environment and makes decisions about whether or not to express a gene, in this case fbp1, which encodes an enzyme. What they found was a preliminary transcription phase with a flurry of switches flicked "on" and then "off" as seen by the synthesis of non-coding RNA before the final "on" switch is tripped.

The non-coding RNAs initiate over one thousand base pairs of nucleotides along the DNA away from the known start site for this gene. The group discovered that the process of transcribing non-coding RNAs is required for the eventual production of the protein-encoding RNA. The transient synthesis of these non-coding RNAs serves to unfurl the tightly wound DNA, essentially loosening the structure to allow for gene expression. [Emphasis added.]

And here's the research article, with some of the abstract, providing a somewhat more precise description of what's going on:

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs

Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1⁺ locus during transcriptional activation. The chromatin at fbp1⁺ is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1⁺. This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1⁺ transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

To expand on that just a bit, recall that chromatin is the form in which DNA is actually stored for safe keeping. It consists of the double-stranded DNA molecules wrapped around many protein complexes called nucleosomes. Before any stretch of DNA can actually be transcribed into messenger RNA, the DNA has to be unwound from the nucleosomes. The present research has determined that some long ncRNA takes part in this unwinding process.

Tags: non-coding RNA, junk DNA, gene expression

Cancer and Myc

Myc is a gene that has been studied for over 20 years and is found to be overexpressed in many human cancers. The protein it codes for (which for simplicity we'll also refer to as Myc) is a transcription factor, like members of the FoxO family, which means it can enable or disable the expression of many other genes. How many? The estimate is about 15% of all human genes. Since there are at least 20,000 genes, the number regulated by Myc is 3000 or more. That's a lot of leverage.

Obviously, Myc is essential for cell function or it wouldn't have so much influence. The biological functions Myc affects include cell proliferation, cell growth, apopotosis, cell differentiation, and stem-cell self-renewal. It is possible for a single transcription factor to have such varied effects because other transcription factors (coactivators or corepressors) must also be present in order to activate or repress the expression of some specific other gene. That is, the expression of any particular gene depends on the presence (or absence) of a particular set of transcription factors.

Since Myc affects the functions mentioned above, it's hardly surprising to find that Myc is overexpressed in many cancers. In such cases, the difference between a normal cell and a cancer cell is the overexpression of Myc in the latter and possibly the abnormal presence of other coactivators or corepressors. The result in a cancer cell is excessive proliferation and/or avoidance of cell death by apoptosis.

Surely you've wondered why development of cancer therapies has taken so long. A large part of the reason is that so many genes involved in cancer interact with so many other genes that have essential functions. So it's not possible to interfere with the cancer-related genes without disrupting vital biological functions elsewhere in the body.

The expression of Myc itself is triggered by signals that are usually external to the cell and normally serve to stimulate cell growth and proliferation when needed, as in lymphocyte production, normal growth, or wound healing. Such signals are called mitogens, because they can initiate mitosis. Wnt, Shh, and EGF are mitogens that can lead to Myc expression.

It is probably almost impossible to hope to combat cancer by directly affecting Myc or its related mitogens, because all of these have essential normal functions themselves. Instead, anti-cancer research needs to examine how Myc may be overexpressed or complemented in harmful ways by other transcription factors.

One promising line of research involves cancer stem cells – cells that, like normal stem cells, can proliferate and differentiate into other cell types, and that also carry cancer-causing mutations. There is now thought to be a "signature" consisting of 11 genes that may be characteristic of cancer stem cells. One of these genes has an effect on Myc:

Scientists Uncover Role Of Cancer Stem Cell Marker: Controlling Gene Expression (1/18/08)

Scientists at Jefferson's Kimmel Cancer Center in Philadelphia have made an extraordinary advance in the understanding of the function of a gene previously shown to be part of an 11-gene "signature" that can predict which tumors will be aggressive and likely to spread. The gene, USP22, encodes an enzyme that appears to be crucial for controlling large scale changes in gene expression, one of the hallmarks of cancer cells. ...

In one example, they looked at the relationship between MYC and USP22. MYC, which is among the most commonly overexpressed genes in cancer, encodes a protein that controls the expression of thousands of other genes. The scientists showed that USP22 is a critical partner of MYC and that by depleting cells of USP22, they could prevent MYC from working properly, stopping it from inducing the invasive growth of cancer cells.

It turns out that Myc affects not only 3000 or so ordinary genes, but also a number of DNA sequences that code for microRNA – and some of these microRNAs play an important role in suppressing cancer. In fact, Myc can stop the production of at least 13 microRNAs:

Silencing Small But Mighty Cancer Inhibitors (12/12/07)

Researchers from Johns Hopkins and the University of Pennsylvania have uncovered another reason why one of the most commonly activated proteins in cancer is in fact so dangerous. As reported in Nature Genetics recently, the Myc protein can stop the production of at least 13 microRNAs, small pieces of nucleic acid that help control which genes are turned on and off.

Furthermore, additional observations showed that some of these microRNAs have an inhibiting effect on cancer, a striking result in itself:

[I]n several instances, re-introducing repressed miRNAs into Myc-containing cancer cells suppressed tumor growth in mice, raising the possibility that a sort-of gene therapy approach could be effective therapy for treating certain cancers.

Since the microRNAs repressed by Myc affect many other genes, there could be thousands of other genes indirectly affected by Myc:

"This study expands our understanding of how Myc acts as such a potent cancer-promoting protein," says Mendell. "We already knew that it can directly regulate thousands of genes. Through its repertoire of miRNAs, Myc likely influences the expression of thousands of additional genes. Activation of Myc therefore profoundly changes the program of genes that are expressed in cancer cells."

More: Researchers zero in on the tiniest members in the war on cancer (12/13/07)

Myc is actually a member of a family of genes, the Myc family. Most members of the family have similar functions. Separate members undoubtedly appeared in the course of evolution from the duplication of earlier members and later mutation. One member, called N-myc, plays a role in both normal development of the retina, as well as cancers of the retina (retinoblastoma).

Recent research has shown that N-myc seems to be responsible for the surprising fact that the retinas in all vertebrates have about the same thickness, regardless of the size of the entire animal or its eyes:

Eye Cancer Gene's Role In Retinal Development Defined (1/18/08)

"A series of complex developmental processes must be carefully orchestrated for the eye to form correctly," said Michael Dyer, Ph.D., associate member in the St. Jude Department of Developmental Neurobiology. "One important aspect of this coordination is that retinal thickness be the same, irrespective of eye size. For example, the mouse eye is about 5,000 times smaller than that of the elephant eye, but the retinal thickness in these two species is comparable."

Working with mice, the researchers found that a gene called N-myc coordinates the growth of the retina and other eye structures to ensure the retina has the proper thickness necessary to convert light from the lens into nerve impulses that the brain transforms into images.

Tags: cancer, Myc, microRNA

Gene activation by CREB

Here's more illustration, if any were needed, of the point that gene expression is a much more complex process than has sometimes been supposed. We recall that transcription factors are proteins that are necessary for a gene to be expressed, and they do their job by binding to a section of DNA (called a promoter) near the gene. To make matters more interesting, it is usually necessary to have other proteins involved, and some bind to the transcription factor rather to the promoter. Such proteins are called cofactors. If the effect is to increase gene expression, the protein is called a coactivator. You may recall that a coactivator played an important role in this post about metering gene expression.

As it happens, we mentioned an important type of transcription factor, called cAMP responsive element binding proteins (CREB) in our recent post on histone deacetylase enzymes. The general situation is that a CREB is part of a cell-signaling pathway, in which a signal arrives at a cell surface, activating some cell surface receptor. As a result, a secondary signal is generated within the cell, consisting of a cAMP molecule, which in turn activates a protein kinase, which finally activates a CREB protein. This last may then act as a transcription factor, causing a particular gene to be expressed, with the resulting protein being the cell's response to receipt of the original signal.

The interesting thing is that, according to the following research, the target gene "chooses" which cofactors are needed along with the CREB to initiate gene transcription. (Is that really surprising? I don't know. It seems one might have guessed that each gene promoter requires something slightly different to allow transcription, so that transcription depends not only on the received signal, but also on other varying conditions.)

Genes Play An Unexpected Role In Their Own Activation, Study Shows

Investigators at St. Jude Children's Research Hospital have discovered how a single molecular "on switch" triggers gene activity that might cause effects ranging from learning and memory capabilities to glucose production in the liver.

The "on switch," a protein called CREB, is a transcription factor--a molecule that binds to a section of DNA near a gene and triggers that gene to make the specific protein for which it codes. CREB activates genes in response to a molecule called cAMP, which acts as a messenger for a variety of stimuli including hormones and nerve-signaling molecules called neurotransmitters.

The St. Jude team showed that each gene that responds to CREB chooses which co-factors, or helper molecules, CREB uses to activate that gene. This finding adds an important piece to the puzzle of how cells use CREB to activate specific genes in response to cAMP signals.

One of the report authors, Paul Brindle, offers this analogy:

"CREB is like a plumber who turns on the water flow in a pipe system by using a certain tool," Brindle said. "What we discovered is that the CREB 'plumber' requires different tools to turn on different genes; and that each gene determines which set of co-factor tools from CREB's toolbox it will respond to."

Tags: CREB, transcription factor, signal transduction, gene expression

Why stem cells are stem cells

This seems like a pretty fundamental finding regarding what makes a stem cell a stem cell:

Five Genetic Themes Key To Keeping Stem Cells In A Primitive, Flexible State Have Been Identified

For more than 25 years, stem cells have been defined based on what they can become: more of themselves, as well as multiple different specialized cell types. But as genetic techniques have become increasingly powerful, many scientists have sought a more molecular definition of stem cells, based on the genes they express.

Now, a team of Canadian scientists has identified 1,155 genes under the control of a gene called Oct4 considered to be the master regulator of the stem cell state. A comprehensive molecular definition of stem cells is emerging: according to this research, stem cells are cells that keep their DNA packaged in a flexible format, keep cell division tightly controlled, prevent signals that might trigger death, repair DNA very effectively, and reinforce all of these characteristics by tightly controlling how molecules can move within the nucleus.

Tags: stem cells

Histone deacetylase enzymes

You don't often hear histone deacetylase (HDAC) enzymes being discussed in ordinary conversation at cocktail parties or around the water cooler – unless perchance you stumble into a conversation among biomedical researchers.

But that might change a bit sometime in the not too distant future. HDAC and HDAC inhibitors are increasingly one of the "hot" topics in cancer research, and their importannce is now leaking out into a variety of disparate areas of biomedicine. There are even connections with other trendy topics, such as the SIR2 "longevity gene" and the NF-κB transcription factors.

Perhaps I should back up a moment and say a few words about histones and histone deacetylases. As you know, DNA generally does not float around all by itself inside a cell. With about 3 billion base pairs, human DNA, simply in order to fit into a cell in an orderly way, needs to be kept most of the time in a very compact form within the 23 chromosome pairs. The material making up chromosomes is called chromatin, which is made up of protein complexes called nucleosomes, around which the DNA is wound. Each nucleosome in turn is made up mostly of a core containing 8 histone proteins of several different types.

This arrangement has important implications for gene expression, because genes that occur in a portion of DNA that is wrapped tightly around a nucleosome are not readily available for translation into messenger RNA, which determines when and how proteins corresponding to the gene can be constructed. However, when an acetyl group is attached to one or more histones of a nucleosome, the DNA becomes less tightly bound, so that its genes can be more easily expressed.

Conversely, removing acetyl groups that may be attached to histones of the nucleosome largely inhibits access to the genes, effectively "silencing" them. A histone deacetylase is an enzyme that removes acetyl groups, so it is a mechanism for silencing groups of genes. About 11 HDACs (depending on how one counts) are known in higher eukaryotic cells.

This gene silencing is anything but a trivial function. For example, the proteins Sirtuin and Sir2 (Sirt1 in mammals), variations of which are found in most eukaryotic cells and are known to be involved with aging, are HDACs. On the other hand, cancer tumors frequently take advantage of HDACs to silence genes that would otherwise promote cancer cell death.

Because of the role of HDACs in cancer, an inhibitor of an HDAC is a potential anti-cancer drug. As we will see, there are a number of these now in clinical trials to fight various cancers – and one has even been approved by the FDA for use (Vorinostat, also known as suberoylanilide hydroxamic acid (SAHA)).

For a great source of technical information on HDACs, especially in relation to cancer, check here.

Following are some research announcements pertaining to HDACs. They are mostly recent, and have been appearing at an increasing rate. Note how some of the most recent ones are in areas well outside of oncology.

Future Therapies For Stroke May Block Cell Death (6/14/07)

Substantial neurological damage occurs in strokes and neurodegenerative diseases like ALS, Parkinson's, and Huntington's. Researchers suspect that there are neuroprotective proteins whose expression could be increased to limit cell death if an inhibitor for the appropriate HDAC can be found.

At Penn, 'tantalizing' finds in cell research (6/14/07)

There are various neurodegenerative diseases which involve damaged or misfolded proteins that are toxic to cells. There is a mechanism called autophagy that is capable of disposing of such proteins, but it does not work fast enough in the presence of disease. Researchers have found that HDAC6 can facilitate autophagy and mitigate disease in a fruit fly model.

Gene Switched Off In Cancer Can Be Turned On, Researchers Discover (6/12/07)

A gene whose protein controls cell growth, called Brahma or BRM, is silent but not missing or mutated in some cancer cells. It is turned off in about 15 percent of tumors studied, including cells from lung, esophageal, ovarian, bladder, colon and breast cancers. HDAC inhibitors were found that could undo the silencing of the gene.

Cancer Drug Enhances Long-term Memory (6/6/07)

In a study with mice, researchers have shown that HDAC inhibitors together with a protein called CBP can enhance memory and actually strengthen neural connections in the hippocampus. CBP is known to relax chromatin, making gene expression easier in affected portions of DNA. Presumably the HDAC inhibitors prevent undoing the effect of CBP. Use of HDAC inhibitors alone did not have an effect on memory. If this effect exists in humans, CBP and HDAC inhibitors could be a therapy for people with Alzheimer's and Huntington's diseases and Rubenstein-Taybi syndrome.

Vorinostat Shows Anti-cancer Activity In Recurrent Gliomas (6/5/07)

Vorinostat is the first FDA approved oral anti-cancer agent that is an HDAC inhibitor. It has been shown to be effective as a treatment for cutaneous T cell lymphoma. This study indicates it also shows activity in patients with recurrent glioblastoma multiforme.

Eat Your Broccoli: Study Finds Strong Anti-Cancer Properties In Cruciferous Veggies (5/18/07)

Cruciferous vegetables such as broccoli, bok choy, and brussels sprouts contain significant amounts of sulforaphane, which has noteworthy anti-cancer properties. This research suggests that cruciferous vegetables have HDAC inhibiting effects, which might explain their anti-cancer properties.

Healthy Muscles: Scientists Identify Pathway That Promotes Muscle Cell Survival In Mice (5/1/07)

Mice genetically engineered with a defective protein called cAMP responsive element binding protein (CREB) have poorly developed muscles. This appears to be related to lack of inhibition of a specific HDAC enzyme when CREB is defective. Investigation revealed that production of an enzyme called salt-inducible kinase-1 (SIK1) is also inhibited in the presence of defective CREB. SIK1 was found to phosphorylate the HDAC protein, which inhibits its histone deacetylation capability. Further experimentation showed that raised SIK1 levels or use of other inhibitors of the HDAC enzyme also restored muscle cell health in the mice with defective CREB. The findings may lead to treatments for diseases that affect cell survival, such as muscular dystrophy, neurodegenerative diseases, and congestive heart failure.

Novel Drug Shows Potential For Treating Leukemia (4/21/07)

HDAC inhibitors, when used in combination with an experimental proteasome inhibitor drug, NPI-0052, were more effective at inhibiting the main enzymatic activity of the proteasome than NPI-0052 alone. Either alone or in combination NPI-0052 was much more effective than bortezomib (marketed as Velcade), the only FDA-approved proteasome inhibitor. Proteasomes clean out mutated or damaged proteins within cells, but in cancer cells this allows unwanted cell growth and reproduction. Proteasome inhibitors block this process, resulting in apoptosis of the malignant cells. Although bortezomib is effective for treating multiple myeloma and mantle cell lymphoma, it is ineffective by itself against leukemia, so NPI-0052 may be a good alternative.

Scientists Induce Cell Death In Leukemia (4/17/07)

The proteasome inhibitor bortezomib when used in combination with either of two HDAC inhibitors (romidepsin and belinostat) was shown in preclinical tests to be very lethal to cultures of human chronic lymphocytic leukemia cells. Other preclinical and clinical data suggest similar synergistic effects of bortezomib in additional cancer cell types.

Treatment Extends Survival In Mouse Model Of Spinal Muscular Atrophy (2/23/07)

Spinal muscular atrophy (SMA) is the most common severe hereditary neurological disease of childhood and is usually fatal. SMA is caused by mutations in a gene called SMN1. A related gene called SMN2 can sometimes produce the SMN protein, but in very small amounts. A drug called trichostatin A (TSA) is a potent HDAC inhibitor that is an antifungal antibiotic and has been found capable of increasing SMN protein production from the SMN2 gene in a mouse model and in cells from SMA patients. Improved survival was observed in the mouse model of SMA.

Two Drugs May Stabilize Plaques In Atherosclerosis (11/15/06)

An anti-fungal drug and an anti-cancer drug – TSA and SAHA (see references elsewhere in this report) – have been reported to decrease cholesterol deposits in the walls of arteries. In this case, the drugs appear to have an anti-inflammatory mechanism. The two compounds decreased inflammatory proteins produced by macrophages taken from normal mice. Such inflammatory proteins can make atherosclerotic plaque unstable. After the macrophages were treated with either TSA or SAHA, dramatic decreases were measured in LDL and total cholesterol in the macrophages. In addition, the drugs prevented macrophages from turning into foam cells inside arterial walls,

Researchers Make Advances In Attacking Leukemia Cells (10/21/05)

This somewhat older research demonstrated that in leukemia cells, HDAC inhibitors also induce changes in a master regulatory protein known as NF-κB, which is involved in regulation of inflammation, cell survival and many other functions. It was found that NF-κB inhibitors dramatically increased the lethality of HDAC inhibitors in various leukemia cell types. Such inhibitors are the subject of great interest as potential anti-inflammatory agents for use in various disorders, such as arthritis and inflammatory bowel disease. The research suggests they may also be valuable in enhancing the antileukemic efficacy of HDAC inhibitors, which have already shown antileukemic activity on their own.

MIT Researchers Uncover New Information About Anti-Aging Gene (2/18/00)

This is old and now well-known research by Leonard Guarente and associates, showing that an anti-aging gene, called Silent Information Regulator (SIR2), is an enzyme – specifically an HDAC enzyme. As such, SIR2 can silence genes in whole sections of a genome. As cells age, problems such as genome instability and inappropriate gene expression surface as genes that had always been turned off sometimes get turned on. SIR2 may forestall such age-related problems, which can lead to cell death. The research team found that yeast cells with an extra copy of SIR2 live longer, while yeast cells without SIR2 have a shorter lifespan. The connection with metabolism (and hence caloric restriction) may stem from a co-enzyme, called nicotinamide adenine dinucleotide (NAD), that is related to metabolism and is required for SIR2 to be activated.

Tags: histone deacetylase, cancer, inflammation, molecular biology, gene expression.

Metering gene expression

As often noted, such as here, gene expression is really quite a complicated process.

Although the net result of DNA transcription is the production of messenger RNA under control of a complex enzyme called RNA polymerase, there's a lot more to it than that. In particular, transcription will usually not even begin unless certain proteins, called transcription factors, have attached themselves to a location on the DNA specific to each gene (called a promoter region). Such proteins are called activators because of the role they play.

To make matters even more complicated, sometimes additional proteins, called coactivators, must be attached to other activators instead of the DNA itself. Since transcription factor proteins are produced under control of other genes, this complex process makes it possible for certain genes to control or regulate the expression of other genes. Indeed, this is the normal state of affairs, and it works much like a computer program, in which the "final" result depends heavily on what else is going on at the same time or earlier.

It now appears that there is a further complication, and hence a further sort of control that is possible. In general it is not desirable that any particular gene remain "on" indefinitely, capable of directing the production of its corresponding protein without limits. Just as with a prescription medicine (which, in some sense, many of the proteins encoded by genes really are), it is often best to dispense only a certain limited quantity. This quantity may be sufficient for whatever its purpose is, and the system may need time to absorb it, with the possibility of producing more later if, and only if, the need still exists.

Research now indicates that in order to allow for such metered usage, some coactivators make it possible to keep a count of how often they are used, and they will automatically be destroyed after the maximum allowed number of uses is reached.

Clocking In And Out Of Gene Expression

"Inherent to the structure of these coactivators is a clock," he said. "But the clock needs to be set off." In studies of breast cancer cells, [senior investigator Dr. Bert] O'Malley and his colleagues showed how the clock works. Using steroid receptor coactivator-3 (SRC-3), they demonstrated that activation requires addition of a phosphate molecule to the protein at one spot and addition of an ubiquitin molecule at another point. Each time the message of the gene is transcribed into a protein, another ubiquitin molecule is chained on. Five ubiquitins in the chain and the protein is automatically destroyed.

"It's built-in self destruction," said O'Malley. "It prevents you from activating a potent factor in the cells that just keeps the clock running and the gene continuing to be expressed." In that scenario, the result could be cancer, too much growth or an abnormal function.

"It means there's a fixed length of time that the molecule can work. When it's activated, it's already preprogrammed to be destroyed. The clock's running and each time an ubiquitin is added, it is another tick of the clock." When the clock system fails, problems result.

Tags: gene expression, molecular biology, gene transcription, transcription factors

RNA tails and gene expression

Only a few years ago – definitely less than ten years – gene expression was thought to be a fairly simple process. One gene coded for one protein. The gene was "transcribed" from DNA to messenger RNA (mRNA), and in turn the mRNA was used to direct the manufacture of proteins in structures called ribosomes.

But then there were a series of "complications". Genes could be turned "on" or "off" by means of transcription factors, which are separate proteins produced by separate genes, and which are capable of either promoting or suppressing the transcription of other genes. Further, genes are not straight uninterrupted segments of DNA that correspond directly (via mRNA) to proteins, because genes contain segments called introns that are edited out of finished mRNA and ignored. And what is more, coding segments of genes (called exons) can be spliced together in different ways to produced finished mRNA (discussed here). This makes it possible to obtain multiple distinct proteins from a single gene.

And then, outside of the RNA transcription process, it turns out that small bits of RNA, called microRNA (miRNA) and small interfering RNA (siRNA), and which are coded for in parts of the genome long thought to be "junk", can become attached to mRNA and inhibit (or perhaps at times promote) production of proteins from it. (See this.) Nor should we forget to mention ribozymes, which can also mess around with mRNA. And if all that weren't enough, there are also a variety of epigenetic factors which can turn on or off entire segments of a genome.

Is that all? No. There are probably a number of other mechanisms that modify, regulate, and control gene expression – mechanisms as yet undiscovered. After all, there's a lot of "junk" DNA, whose function we still have no clue about – except that a lot of it isn't truly "junk".

Here's an example that has just come to light: RNA "tails".

Yeast: The Key To Understanding How Cells Work

The major contribution to the collaborative study by Associate Professor Preiss' Lab was to measure the length of polyadenosine "tails" on the messenger RNA (mRNA) molecules that are generated from each gene to serve as a blueprint in making proteins - the building blocks of life.

"One might think that a tail does not matter much, but with mRNAs it has a big impact on how long they stay around in cells and how much protein is made from them. In this way it is a case of the tail wagging the dog. Since nearly every mRNA in every human cell has these tails, it is not surprising that controlling their length turns out to be quite important. It is known to be involved in embryonic development and during learning and memory in the brain, for instance. The mRNA tails also seem to be the target for recently discovered tiny cellular brakes called microRNAs. Failure of these brakes contributes to human diseases, such as heart defects and cancer.

Tags: RNA tails, molecular biology, gene expression

P53 protein and tanning

At first this may seem an odd coincidence, but maybe it isn't. P53 is the protein which plays a critical role in preventing runaway division in cells with damaged DNA, and hence inhibiting cancer. Unless p53 itself becomes faulty – which happens in the majority of cancerous cells. It does its job by stopping the cell division cycle if damaged DNA is detected.

But apparently p53 is also implicated in tanning of human skin by the sun.

'Guardian Of The Genome' Protein Found To Underlie Skin Tanning

A protein known as the "master watchman of the genome" for its ability to guard against cancer-causing DNA damage has been found to provide an entirely different level of cancer protection: By prompting the skin to tan in response to ultraviolet light from the sun, it deters the development of melanoma skin cancer, the fastest-increasing form of cancer in the world.

In a study in the March 9 issue of the journal Cell, researchers at Dana-Farber Cancer Institute report that the protein, p53, is not only linked to skin tanning, but also may play a role in people's seemingly universal desire to be in the sun -- an activity that, by promoting tanning, can reduce one's risk of melanoma.

"The number one risk factor for melanoma is an inability to tan; people who tan easily or have dark pigmentation are far less likely to develop the disease," says the study's senior author, David E. Fisher, MD, PhD, director of the Melanoma Program at Dana-Farber and a professor in pediatrics at Children's Hospital Boston. "This study suggests that p53, one of the best-known tumor-suppressor proteins in our body, has a powerful role in protecting us against sun damage in the skin."

Of course, people who tan easily or have dark pigmentation may also be less inclined to spend time in the sun for the purpose of acquiring a tan, so any other factors in an individual that might be responsible for tannning or dark pigmentation would also indirectly reduce the statistical liklihood of melanoma.

However, the research shows that p53 does influence tanning directly.

Other reports:

• Gene behind tanning comes out of hiding
  
• A Protein Twofer That Triggers Tanning and Protects against Skin Cancer
  
• Anti-Cancer Gene Triggers Tanning
  

Update 8/3/08: There is related news about this here.

Tags: p53, sun tanning, melanoma, skin cancer, cell cycle

MicroRNA

MicroRNA (miRNA) is a short (about 21 to 23 nucleotides) single-stranded RNA molecule that is now recognized as playing an important role in gene regulation – even though the term has been in use only since 2001. It is similar to, but distinct from, another type of short RNA, known as small interfering RNA (siRNA).

Although miRNA and siRNA both have gene regulation functions, there are subtle differences. MiRNA may be slightly shorter than siRNA (which has 20 to 25 nucleotides). MiRNA is single-stranded, while siRNA is formed from two complementary strands. The two kinds of RNA are encoded slightly differently in the genome. And the mechanism by which they regulate genes is slightly different.

MiRNA attaches to a piece of messenger RNA (mRNA) – which is the master template for building a protein – in a non-coding part at one end of the molecule. This acts as a signal to prevent translation of the mRNA into a protein. SiRNA, on the other hand, attaches to a coding region of mRNA, and so it physically blocks translation.

In addition to the Wikipedia articles, here's another handy source of information on miRNA.

There have been several research results reported recently that illustrate some of the important functional roles of miRNA.

MicroRNA and cancer

The importance of gene regulation by miRNA is not trivial. As the following article notes, "microRNAs found in mammals regulate over a third of the human genome, as shown in a 2005 study by the lab of Whitehead Member and Howard Hughes Medical Institute Investigator David Bartel and colleagues." (Reference: MicroRNAs Have Shaped The Evolution Of The Majority Of Mammalian Genes)

Since either overexpression or underexpression of certain genes can cause cancer, it's not surprising that miRNA should have significant cancer-related effects.

MicroRNA helps prevent tumors

Looking to find a promising target for an individual microRNA, Christine Mayr, a postdoctoral researcher in the Bartel lab, picked Hmga2, a gene that is defective in a wide range of tumors.

In these tumors, the protein-producing part of the Hmga2 gene is cut short and replaced with DNA from another chromosome. Biologists have mostly focused on the shortened protein as the possible reason that the cells with this DNA swap became tumors. But this DNA swap removes not only the gene's protein-producing regions but also those areas that don't code for protein. And these non-protein-producing regions contain the elements that microRNAs recognize.

It turns out that in the non-protein-producing region, Hmga2 has seven sites that are complementary to the let-7 microRNA, a microRNA expressed in the later stages of animal development. Mayr wondered whether loss of these let-7 binding sites, and therefore loss of regulation by let-7 of Hmga2, might cause over-expression of Hmga2 that in turn would result in tumor formation.

This turned out to be a very good guess:

Overall, the results highlight a new mechanism for cancer formation. Hmga2, and perhaps certain other genes that are normally regulated by microRNAs, can help give rise to tumors if a mutation in the gene disrupts the microRNA's ability to regulate it.

MicroRNA and stem cells

It's not news to anyone that research into stem cells is a very active area these days. It turns out that miRNA may play a key role in keeping stem cells from differentiating prematurely into normal body cells.

Master Switches Found For Adult Blood Stem Cells

Johns Hopkins Kimmel Cancer Center scientists have found a set of "master switches" that keep adult blood-forming stem cells in their primitive state. Unlocking the switches' code may one day enable scientists to grow new blood cells for transplant into patients with cancer and other bone marrow disorders.

The scientists located the control switches not at the gene level, but farther down the protein production line in more recently discovered forms of ribonucleic acid, or RNA. MicroRNA molecules, once thought to be cellular junk, are now known to switch off activity of the larger RNA strands which allow assembly of the proteins that let cells grow and function.

Since a miRNA molecule can attach itself to a mRNA molecule if there is a match in only about seven consecutive nucleotides, it wouldn't be surprising if one miRNA could regulate the translation of many different proteins. And indeed, this is one of the findings of the research:

To identify the key microRNAs, Georgantas sifted through thousands of RNA pieces with a custom-built, computer software program. Its algorithms let the software, fed data from samples of blood and bone marrow from healthy donors, match RNA pairs. The outcome was a core set of 33 microRNAs that match with more than 1,200 of the larger variety RNA already known to be important for stem-cell maturation.

Just as important for the persistence of a species are stem cells for germline cells – eggs and sperm.

MicroRNA Pathway Essential For Controlling Self-renewal Of Stem Cells

"The findings were interesting to us because they demonstrated that the microRNA pathway is essential for controlling self-renewal or maintenance of two types of stem cells – germline stem cells and somatic stem cells," said Dr. Jin. "In the future, the small RNAs responsible for stem cell regulation could potentially be used to control stem cell functions in vivo and stem cell expansion in vitro."

Editing of microRNA

We have noted that a single miRNA can affect the expression of a large set of genes. It turns out that relatively minor editing of the miRNA after initial transcription can cause it to affect a completely different set of genes:

Killing the messenger RNA — But which one?

Now, a new study led by researchers at The Wistar Institute shows that these microRNAs can undergo a kind of molecular editing with significant physiological consequences. A single substitution in their sequence can redirect these microRNAs to target and silence entirely different sets of genes from their unedited counterparts. Further, errors in the editing can lead to serious health problems.

"What we found was that, in certain cases, edited versions of these microRNAs are being produced that differ from the unedited versions by only a single nucleotide change," says Kazuko Nishikura, Ph.D., a professor in the Gene Expression and Regulation Program at Wistar and senior author on the study. "These edited microRNAs are not encoded in the DNA, which means that at least two versions can being produced by one gene.

If there's one conclusion to be derived from all this recent research, it is that a relative handful of miRNA species – several hundred have been identified so far, compared with 25,000 or so protein-coding genes in humans – are capable of drastically influencing all kinds of cellular processes. That's a lot of "leverage". Applied at the right times and places, miRNA could provide therapies for a large number of diseases. But of course, the ability of a single miRNA to affect so many different genes means that they have to be targeted very, very carefully. It will be interesting to see how this plays out.

Tags: MicroRNA, miRNA, RNA interference, RNAi, stem cells, cancer

Holiday gifts for science folks

Sorry to mention these so late, but I just came across this site – Bathsheba Sculpture – from a talented artist. I won't violate the artist's copyright by putting some pictures here or hotlink them – just have a look at the site. The artist, Bathsheba Grossman, does sculptures of mathematical forms using 3D printing technology, and also creates images laser-etched inside glass. The technology is quite interesting too.

For folks who are more into biology than math, the artist also does protein models etched in glass, shown at her other site: Crystal Protein. If you happen to have a PDB code for a favorite protein of yours, you can even get a custom model made.

I don't know whether Bathsheba's work is unique, as I'm not very familiar with the art world. All I can say is that I'd certainly feel proud to give this kind of gift, or surprised and fortunate to receive such.

Tags: mathematical sculpture, protein models, art

RNA activation of genes

The subject of RNA has come up in a number of scientific developments recently. It seems that RNA occurs in more forms and plays more roles within cells than scientists have previously supposed.

Some of the important forms that RNA can take have been known for some time. The oldest of these are messenger RNA (mRNA), which is an intermediate stage in the translation of genetic information from DNA to proteins, and transfer RNA (tRNA), which assists in the making of proteins in a ribosome. Further, ribosomes themselves are made up of some proteins and another type of RNA, ribosomal RNA (rRNA). Besides that, RNA is the genetic material of the type of viruses known as retroviruses, which include HIV.

In the 1980s, forms of RNA, called ribozymes, that act as catalysts in cellular chemistry, were discovered – and the discovery led to a Nobel Prize.

All of the forms of RNA found in cells, except for tRNA, are known collectively as non-coding RNA (ncRNA) because they do not directly encode the information in genes. Within the past 10 years a number of additional types of ncRNA have been found, including microRNA (miRNA) and small interfering RNA (siRNA).

Small interfering RNA is a big deal, big enough that the discovery has already lead to the awarding of a Nobel prize this year, though the discovery occurred less than 10 years ago:

Nobel prize for genetic discovery

Two US scientists have been awarded the Nobel Prize for medicine for their pioneering work in genetics.

The work of Dr Andrew Fire and Dr Craig Mello could lead to new treatments for a range of illnesses, including viral infections and cancer.

They discovered a phenomenon called RNA interference, which regulates the expression of genes.

The process has the potential to help researchers shut down genes which cause harm in the body.

The breakthrough has also given scientists the ability to systematically test the functions of all human genes.

The process by which siRNA can interfere with the expression of certain genes is known as RNA interference (RNAi). The process can occur by at least two mechanisms and has been thoroughly verified.

Now there is a surprizing, and controversial, claim that similar short RNA molecules can boost the expression of some genes:

How to get your genes switched on

The latest twist on the Nobel prizewinning method of RNA interference, or RNAi, could prove to be a real turn-on. Whereas standard RNAi silences a target gene, switching protein production off, the new technique boosts gene activity, providing a genetic "on" switch.

RNAi can silence genes in two ways. It can block the messenger RNA that is the intermediate between gene and protein and it can also interfere with "promoter" sequences that boost a gene's activity. It was while investigating this second phenomenon that Long-Cheng Li of the University of California, San Francisco, and his colleagues stumbled on the new method, dubbed RNA activation.

There's more detail in this article from Science (subscription rqd):

Small RNAs Reveal an Activating Side

This surprising skill--dubbed RNAa, because the RNAs activate genes--is described this week in the online edition of the Proceedings of the National Academy of Sciences. If the claim is sustained, RNAa would be a powerful biological tool and could lead to new therapies for diseases such as cancer. But some scientists say the results may reflect an indirect outcome of RNAi, rather than a new way to activate genes. "It's going to be a question of whether this holds up," says Erik Sontheimer, an RNA researcher at Northwestern University in Evanston, Illinois.

At this point, it seems that the gene activation could occur because the production of an inhibitory protein is blocked by conventional RNAi.

One key question is whether Li's RNAs are activating genes by silencing others, which would just be RNAi by another name. For example, proteins called negative transcription factors can prevent genes from being transcribed; silencing the genes for these proteins could activate genes they control.

But there is evidence that something different might be happening.

No one yet knows how small RNAs could turn genes on, especially for so long. RNAi typically silences genes for 5 to 7 days, but RNAa boosted gene activity for up to 13 days. The molecular machinery underlying RNAi appears to be involved in RNAa, raising the question of how the same enzymes can sometimes turn genes off, and sometimes on. "What makes one siRNA [small interfering RNA] a silencer, and what makes the other one an activator?" asks Sontheimer. "No clue."

Additional information:

Small dsRNAs induce transcriptional activation in human cells – original research paper (subscription rqd for full access)

Tags: biology, molecular biology, RNA, gene regulation, RNA interference, RNA activation

Alternative splicing

Not so very long ago it used to be that molecular biologists thought that for every protein in the body there was a specific gene, and every gene contained the instructions for making just one protein. Then, when the human genome was completely mapped several years ago, it was found, to everyone's embarrassment, that there were a lot fewer than 25,000 different genes in the genome. This is in a genome of 3.12 billion base pairs. And the human genome is far from the largest. Ordinary corn has 5 billion base pairs and 50,000 genes. The trumpet lily plant (Lilium longiflorum) has 90 bilion base pairs in its genome, and the marbled lungfish (Protopterus aethiopicus) has 139 billion – but apparently nobody has had the patience to sit down and count their actual genes. (Reference; see also here.)

Anyhow, it's estimated that humans use at least 100,000 different proteins, maybe a lot more, so the point is that some genes must be capable of coding for a lot more than just one protein. It's now understood that this is accomplished by the process known as alternative splicing. As you know, genes are not simple, uninterrupted sequences of base pairs. They have within them several subsequences known as exons and introns. In a nutshell, the exons are eventually transcribed into messenger RNA, while the introns are discarded.

Except there's a little more to it than that. In order to produce different proteins, it's necessary to select a subset of exons to code for each particular protein. So how does this actually happen? Some new research has figured this out in one specific case:

RNA Map Provides First Comprehensive Understanding Of Alternative Splicing

It's biology's version of the director's cut. In much the same way that numerous films could be stitched together from a single reel of raw footage, a molecular process called alternative splicing enables a single gene to produce multiple proteins. Now a new RNA map, created by a team of researchers at Rockefeller University and the Howard Hughes Medical Institute and announced in the journal Nature, shows for the first time how the specific location of short snippets of RNA affects the way that alternative splicing is controlled in the brain.

Though scientists have begun to appreciate how alternative splicing adds a layer of complexity to brain processes that enable us to think and learn, exactly how alternative splicing is regulated during these processes -- and in some cases is uncontrolled (or dysregulated) to cause disease -- has remained elusive. The map provides the first comprehensive understanding of how alternative splicing works throughout the genome. The results have implications for a better understanding of such brain functions as learning and memory, neurological diseases and cancer biology.

To make a long story short, there is a brain protein called Nova that was known to be capable of binding to 50 different sequences of RNA. The study found that there were actually 30 different exons which contained those sequences, and whether or not a given sequence had been bound by Nova could cause the exon to be either included or excluded (depending on circumstances) from a final transcript.

This is of more than just theoretical interest. Errors in the transcription process can cause a variety of disease conditions:

By offering a global understanding of how alternative splicing works across the genome, the map has implications for the treatment of a growing list of human neurologic diseases in which RNA regulation, and particularly RNA splicing, has been implicated as the primary cause, including certain types of cancer and a number of brain and muscle disorders.

"Given that the complexity of the brain is orders and orders of magnitude more complex than the number of genes we have, one of the intriguing things about alternative splicing is that a relatively small number of regulatory splicing factors acting in concert on a single transcript can potentially generate a large number of different protein variants," says Darnell.

"There is a converging set of observations indicating that as neurologic diseases are better understood, alternative splicing is going to play an important role in generation of disease and therefore an important role in normal generation of cognitive function," he adds. "Our new work lays out an approach to developing a global understanding of how alternative splicing is regulated by one disease-associated protein, Nova, offering a route by which scientists may now be able to approach a number of diseases with a fresh start."

It's interesting, also, that this process is being observed in the brain. Because, as Antonio Damasio has just predicted for New Scientist as one of the most likely discoveries of the next 50 years, we should learn how relatively few genes can create such complexity in the brain:

Most of what I regard as exciting in recent neuroscience has concentrated on two broad areas: molecular neurobiology and an understanding of the systems related to cognition and behavior. The future will no doubt promote advances in those two areas. On the molecular side, it will be possible to know how so few genes (relatively speaking) create so much complexity in the human brain.

It would be a good guess that the use of alternative splicing is pretty common in brain tissue.

Update: And in fact, I wrote about this very topic a year ago: RNA splicing occurs in nerve-cell dendrites. The interesting thing is that in most cells, splicing is known to occur only in the nucleus. In neurons, however, it occurs in dendrites, the part of a neuron to which other neurons form connections.

Tags: medicine, biology, alternative splicing