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

P53, a versatile gene

P53 is well-known for its role in regulating the cell cycle so as to suspend the cycle or even lead to cell death via apoptosis in case damage to a cell's DNA is detected. This function is especially important in forestalling cancer.

And as we noted here, p53 is also involved with skin tanning.

But that's not all p53 is good for. It also plays a role in fertility, which has recently been reported by one of the co-discoverers (Arnold Levine) of p53:

Cancer Fighter May Be Fertility Helper

A protein known primarily for its role in fighting cancer also helps embryos implant in the womb, according to a study in mice. The find may explain why some women have difficulty becoming pregnant.

More information: here, here

But the list of p53's goodness doesn't stop there. It also slows aging, apart from deterring cancer, but via the same mechanism:

Anti-cancer gene p53 doubles up as anti-ageing agent

The latest research suggests that one of the genes that protects us from cancer may also help delay the ageing process.

A new study has found that a particular gene, p53 which has been previously linked to premature ageing, along with one of its cellular regulators, called Arf, may boost the body's antioxidant activity to keep cells younger longer and thereby slow down the aging process.

The regulatory chemical Arf, lets p53 know that a particular cell is in trouble and marked for elimination.

More information: here, here, here, here

But, surprisingly, at least in fruit flies, reducing p53 activity may also increase lifespan, and apparently in the same way that calorie restriction does:

Key To Longer Life (in Flies) Lies In Just 14 Brain Cells

Two years ago, Brown University researchers discovered something startling: Decrease the activity of the cancer-suppressing protein p53 and you can make fruit flies live significantly longer.

Now the same team reports an intriguing follow-up finding. The p53 protein, they found, may work its lifespan-extending magic in only 14 insulin-producing cells in the fly brain.

How was this connected with calorie restriction? Simply by noting that calorie restriction in fruit flies didn't increase longevity when p53 activity was suppressed in only 14 insulin-producing cells of the flies' brains:

Studies have shown that low-calorie diets can significantly increase the lifespan of flies, worms, mice and rats. The phenomenon is of intense interest to researchers who study aging. They want to know if caloric restriction works in people and if drugs could be made to mimic its effects.

So researchers restricted the diets of the flies and ran the same experiments. The calorie-restricted flies didn't live any longer when p53 was reduced in the insulin-producing cells. This evidence supports the notion that p53 reduction is one of the direct effects of caloric restriction.

Even more intriguing, Helfand said, is the fact that the 14 insulin-producing cells that seem to be critical for lifespan extension are the equivalent of beta cells in the human pancreas. Beta cells make and release insulin, the hormone that controls the level of glucose in the blood. The research team found that when p53 activity drops, so does insulin-responsive activity in the fat body, the major metabolic organ in the fruit fly.

The involvement of insulin in this effect is especially interesting, as insulin signaling has also been found to be involved in the mechanism by which sirtuin proteins extend longevity in nematodes and fruit flies (and perhaps other organisms).

One wonders just how p53 came to play such a prominent role in cellular processes. Some researchers think they have found the answer – endogenous retroviruses that have actually proven beneficial to the host genome:

Ancient Retroviruses Spurred Evolution Of Gene Regulatory Networks In Humans And Other Primates

Scientists have long wondered how a master regulator such as p53 gained the ability to turn on and off a broad range of other genes related to cell division, DNA repair, and programmed cell death. How did p53 build its complex and powerful empire, so to speak?

Using the tools of computational genomics, the UCSC team gathered compelling evidence that retroviruses helped out. ERVs jumped into new positions throughout the human genome and spread numerous copies of repetitive DNA sequences that allowed p53 to regulate many other genes, the team contends.

"This would have provided a mechanism to quickly establish a gene regulatory network in a very short evolutionary time frame," said Ting Wang, a post-doctoral researcher at UCSC and lead author of the paper.

It's hard to avoid a suspicion that there's a lot to the story of p53 left to be discovered.

Tags: p53, aging, longevity, calorie restriction, insulin signaling

HDAC drugs for autoimmune diseases

Initially it may come as a surprise, but it turns out that some powerful cancer drugs also are useful against autoimmune diseases as well. A good example is the monoclonal antibody rituximab.

In this case the reason is that as a monoclonal antibody, rituximab binds to the protein CD20, which is widely expressed on B cell lymphocytes of the immune system. Once it has bound to the CD20 of a B cell, it may lead to cell death by apoptosis, which is helpful in the case of of B cell cancers such as non-Hodgkin lymphoma and B cell leukemia.

The general pattern here is that some kinds of cancer and some autoimmune diseases both involve the overactivity and proliferation of certain types of immune system cells. A drug that counteracts such pathology may be useful for treating both the associated cancers and autoimmune diseases.

T cells are another large and important class of immune system cells, which we've discussed before (see here). T cells can also be involved in cancer, such as another form of non-Hodgkin lymphoma called cutaneous T cell lymphoma.

There is an FDA-approved drug for this cancer, called vorinostat, also known as suberoylanilide hydroxamic acid (SAHA). We have also mentioned this before, because the compound is a histone deacetylase (HDAC) enzyme inhibitor. HDAC enzymes have the effect of turning off genes, so an inhibitor of a particular HDAC enzyme has the effect of allowing the genes to remain turned on. Some cancers develop because they cause the overexpression of a HDAC enzyme that then turns off genes which would otherwise suppress the cancer. So an inhibitor of the approriate HDAC enzyme boosts the expression of the affected cancer-fighting genes. This is how vorinostat works.

Certain types of T cells can also cause various autoimmune diseases if they get out of control. Normally these T cells should be kept under control by messaging molecules (such as Foxp3) produced by a special type of T cell called a regulatory T cell (T_reg cells). So when a condition develops where there is excessive and harmful activity of T cells, it it may be possible to counteract this by boosting the number or activity of T_reg cells.

And this is precisely what SAHA is apparently able to do – by inhibiting a HDAC enzyme, it raises T_reg cell activity to control the overactivity of other types of T cells, as found in inflammatory bowel disease and various kinds of transplant rejections.

New cancer drugs could help in autoimmune disease

A new class of drugs used to treat cancer might be effective at suppressing overactive immune systems in patients with autoimmune diseases like Crohn's disease, U.S. researchers said on Sunday.

"What we would be proposing would be a therapy that would enhance the body's own immune system's ability to regulate itself," said Wayne Hancock of Children's Hospital of Philadelphia, whose study appears in the journal Nature Medicine.

Hancock said drugs known as histone deacetylases inhibitors, or HDACs, which affect compounds involved in the growth and death of cancer cells, bolstered the production of cells that regulate the immune system in mice.

In one study, the drug helped reverse and prevent inflammatory bowel disease. It also prevented the rejection of heart transplants in other mice. And it stopped rejection of pancreatic cell transplants in other mice.

It's possible that SAHA (i. e. vorinostat, which is distributed commercially by Merck under the name Zolinza) could also work to treat other autoimmune diseases like rheumatoid arthritis. And who knows what other cancer drugs might also have such dual uses?

More: Cancer drugs could fight autoimmune disease

Tags: cancer, autoimmune disease, histone deacetylase enzyme

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

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

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

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

RNA splicing occurs in nerve-cell dendrites

A single gene is capable of directing the production of a number of different proteins. This is because each gene consists of regions ("exons") that code for amino acids that make up a protein, and other regions ("introns") that do not. Initially a gene is transcribed into RNA. But before this RNA is used to construct a protein, the introns are removed in a process called "splicing". Exons may be removed, as well, before the final messenger RNA is complete. Variant forms of the messenger RNA result depending on which exons are removed.

Normally this splicing process occurs in the nucleus of a cell. But new research shows that it can also occur in the parts of nerve cells known as "dendrites", which receive incoming signals from other nerve cells. It may be that the production of variant proteins from a single gene affects the strength of connections between nerve cells, and hence is involved in the processes of learning and memory.

RNA splicing occurs in nerve-cell dendrites

Protein diversity is a key aspect to the complexity of the central nervous system. Proteins are the workhorses of the cell and are generally responsible for insuring that cells function properly. When proteins interact with one another they can elicit specific physiological responses, including the generation and maintenance of memories. Changing protein identity, as can occur with splicing, can change the ability of the protein to interact with other proteins and therefore potentially change such physiological processes. With the dendrite being the initial site in the neuron where learning is thought to occur, the ability to create a diversity of mRNAs, through local splicing, and subsequent protein translation may permit exquisitely sensitive control of these cellular functions.

Tags: RNA splicing