Maybe there really is junk DNA

Until about 20 years ago, molecular biology was incredibly protein-centric.  Consider the following terms — nonsense codon, noncoding DNA, junk DNA.  All are pejorative and arose from the view that all the genome does is code for protein.  Nonsense codon means one of the 3 termination codons, which tells the ribosome to stop making protein.  Noncoding DNA means not coding for protein (with the implication that DNA not coding for protein isn’t coding for anything).

The term Junk DNA goes back to the 60s, a time of tremendous hubris as the grand biochemical plan of life was being discovered. People were not embarrassed to use the term ‘central dogma’ which was DNA makes RNA makes protein. It therefore came as a shock once we had a better handle on the size of the genome to discover that less than 2% of it coded for protein. Since much of it was made of repetitive sequences it was called junk DNA.

I never bought it, thinking it very dangerous to dismiss as unimportant what you did not understand or could not measure. Probably this was influenced by my experience as an Air Force M.D. ’68 – ’70 during the Vietnam war.

But now comes a sure to be contentious but well reasoned paper arguing that junk DNA does exist, even though it is occasionally transcribed [ Cell vol. 183 pp. 1151 – 1161 ’20 ]. The paper discusses all RNAs in the cell not part of the ribosome, or small nucleolar RNAs (snoRNAs) or microRNAs.

They note that no enzyme is perfect acting on only the substrate we think evolution optimized it for — they call this promiscuous behavior. So a transcription factor which binds to a particular promoter sequence will also bind to near miss sequence. Moreover such near misses are constantly being generated in our genome by random mutation. This is why they think that the ENCODE (ENCyopedia Of Dna Elements) found that the entire genome is transcribed into RNA. The implication made by many is that this must be functional.

However many random pieces of DNA can activate transcription [ Genes Dev. vol. 30 pp. 1895 – 1907 ’16 ] producing what the authors call transcriptional noise.

There is evidence that the cell has evolved a way to stop some of this. U1 snRNP recognizes the 5′ splice site motif. It is present in nuclei at an order of magnitude higher than other spliceosomal subcomplexes, so it monitors for RNAs which have a 5′ splice site motif but which lack the 3′ splice site. These RNAs are subsequently destroyed, never making it out of the nucleus.

They think the primary function of lncRNA is chromatin remodeling affecting gene expression — this is certainly true of XIST which silences one of the two X chromosomes females carry.

There is a lot more very technical molecular biology and close reasoning in the paper, but this should be enough to whet your interest. It is well worth reading. Probably, like me, you’ll be mentally arguing with the authors as you read it, but that’s the sign of a good paper.

Now for a question which has always puzzled me. Consider the leprosy organism. It’s a mycobacterium (like the organism causing TB), but because it essentially is confined to man, and lives inside humans for most of its existence, it has jettisoned large parts of its genome, first by throwing about 1/3 of it out (the genome is 1/3 smaller than TB from which it is thought to have diverged 66 million years ago), and second by mutation of many of its genes so protein can no longer be made from them. Why throw out all that DNA? The short answer is that it is metabolically expensive to produce and maintain DNA that you’re not using

If you want a few numbers here they are:
Genome of M. TB 4,441,529 nucleotides
Genome of M. Leprae 3,268,203 nucleotides

Clearly microorganisms are under high selective pressure, and the paper says that humans are under almost none, but it seems to me that multicellular organisms would have found a way to get rid of DNA it doesn’t need.

It may well be that all this DNA and the RNA transcribed from it is evolutionary potting soil, waiting for some new environmental stress to put it to use.

Takes me right back to grad school

How many times in grad school did you or your friends come up with a good idea, only to see it appear in the literature a few months later by someone who’d been working on it for much longer. We’d console ourselves with the knowledge that at least we were thinking well and move on.

Exactly that happened to what I thought was an original idea in my last post — e.g. that Gemfibrozil (Lopid) might slow down (or even treat) Alzheimer’s disease. I considered the post the most significant one I’d ever written, and didn’t post anything else for a week or two, so anyone coming to the blog for any reason would see it first.

A commenter on the first post gave me a name to contact to try out the idea, but I’ve been unable to reach her. Derek Lowe was quite helpful in letting me link to the post, so presently the post has had over 200 hits. Today I wrote an Alzheimer’s researcher at Yale about it. He responded nearly immediately with a link to an ongoing clinical study in progress in Kentucky

On Aug 3, 2015, at 3:04 PM, Christopher van Dyck wrote:

Dear Dr. xxxxx

Thanks for your email. I agree that this is a promising mechanism.
My colleague Greg Jicha at U.Kentucky is already working on this:
https://www.nia.nih.gov/alzheimers/clinical-trials/gemfibrozil-predementia-alzheimers-disease

Our current efforts at Yale are on other mechanisms:
http://www.adcs.org/studies/Connect.aspx

We can’t all test every mechanism, but hopefully we can collectively test the important ones.

-best regards,
Christopher H. van Dyck, MD
Professor of Psychiatry, Neurology, and Neurobiology
Director, Alzheimers Disease Research Unit

Am I unhappy about losing fame and glory being the first to think of it?  Not in the slightest.  Alzheimer’s is a terrible disease and it’s great to see the idea being tested.

Even more interestingly, a look at the website for the study shows, that somehow they got to Gemfibrozil by a different mechanism — microRNAs rather than PPARalpha.

I plan to get in touch with Dr. Jicha to see how he found his way to Gemfibrozil. The study is only 1 year in duration, and hopefully is well enough powered to find an effect. These studies are incredibly expensive (and an excellent use of my taxes). I never been involved in anything like this, but data mining existing HMO data simply has to be cheaper. How much cheaper I don’t know.

Here’s the previous post —

Could Gemfibrozil (Lopid) be used to slow down (or even treat) Alzheimer’s disease?

Is a treatment of Alzheimer’s disease at hand with a drug in clinical use for nearly 40 years? A paper in this week’s PNAS implies that it might (vol. 112 pp. 8445 – 8450 ’15 7 July ’15). First a lot more background than I usually provide, because some family members of the afflicted read everything they can get their hands on, and few of them have medical or biochemical training. The cognoscenti can skip past this to the text marked ***

One of the two pathologic hallmarks of Alzheimer’s disease is the senile plaque (the other is the neurofibrillary tangle). The major component of the plaque is a fragment of a protein called APP (Amyloid Precursor Protein). Normally it sits in the cellular membrane of nerve cells (neurons) with part sticking outside the cell and another part sticking inside. The protein as made by the cell contains anywhere from 563 to 770 amino acids linked together in a long chain. The fragment destined to make up the senile plaque (called the Abeta peptide) is much smaller (39 to 42 amino acids) and is found in the parts of APP embedded in the membrane and sticking outside the cell.

No protein lives forever in the cell, and APP is no exception. There are a variety of ways to chop it up, so its amino acids can be used for other things. One such chopper is called ADAM10 (aka Kuzbanian). ADAM10breaks down APP in such a way that Abeta isn’t formed. The paper essentially found that Gemfibrozil (commercial name Lopid) increases the amount of ADAM10 around. If you take a mouse genetically modified so that it will get senile plaques and decrease ADAM10 you get a lot more plaques.

The authors didn’t artificially increase the amount of ADAM10 to see if the animals got fewer plaques (that’s probably their next paper).

So there you have it. Should your loved one get Gemfibrozil? It’s a very long shot and the drug has significant side effects. For just how long a shot and the chain of inferences why this is so look at the text marked @@@@

****

How does Gemfibrozil increase the amount of ADAM10 around? It binds to a protein called PPARalpha which is a type of nuclear hormone receptor. PPARalpha binds to another protein called RXR, and together they turn on the transcription of a variety of genes, most of which are related to lipid metabolism. One of the genes turned on is ADAM10, which really has never been mentioned in the context of lipid metabolism. In any event Gemfibrozil binds to PPARalpha which binds more effectively to RAR which binds more effectively to the promoter of the ADAM10 gene which makes more ADAM10 which chops of APP in such fashion that Abeta isn’t made.

How in the world the authors got to PPARalpha from ADAM10 is unknown — but I’ve written the following to the lead author just before writing this post.

Dr. Pahan;

Great paper. People have been focused on ADAM10 for years. It isn’t clear to me how you were led to PPARgamma from reading your paper. I’m not sure how many people are still on Gemfibrozil. Probably most of them have some form of vascular disease, which increases the risk of dementia of all sorts (including Alzheimer’s). Nonetheless large HMOs have prescription data which can be mined to see if the incidence of Alzheimer’s is less on Gemfibrozil than those taking other lipid lowering agents, or the population at large. One such example (involving another class of drugs) is JAMA Intern Med. 2015;175(3):401-407, where the prescriptions of 3,434 individuals 65 years or older in Group Health, an integrated health care delivery system in Seattle, Washington. I thought the conclusions were totally unwarranted, but it shows what can be done with data already out there. Did you look at other fibrates (such as Atromid)?

Update: 22 July ’15

I received the following back from the author

Dear Dr.

Wonderful suggestion. However, here, we have focused on the basic science part because the NIH supports basic science discovery. It is very difficult to compete for NIH R01 grants using data mining approach.

It is PPARα, but not PPARγ, that is involved in the regulation of ADAM10. We searched ADAM10 gene promoter and found a site where PPAR can bind. Then using knockout cells and ChIP assay, we confirmed the participation of PPARα, the protein that controls fatty acid metabolism in the liver, suggesting that plaque formation is controlled by a lipid-lowering protein. Therefore, many colleagues are sending kudos for this publication.

Thank you.

Kalipada Pahan, Ph.D.

The Floyd A. Davis, M.D., Endowed Chair of Neurology

Professor

Departments of Neurological Sciences, Biochemistry and Pharmacology

So there you have it. An idea worth pursuing according to Dr. Pahan, but one which he can’t (or won’t). So, dear reader, take it upon yourself (if you can) to mine the data on people given Gemfibrozil to see if their risk of Alzheimer’s is lower. I won’t stand in your way or compete with you as I’m a retired clinical neurologist with no academic affiliation. The data is certainly out there, just as it was for the JAMA Intern Med. 2015;175(3):401-407 study. Bon voyage.

@@@@

There are side effects, one of which is a severe muscle disease, and as a neurologist I saw someone so severely weakened by drugs of this class that they were on a respirator being too weak to breathe (they recovered). The use of Gemfibrozil rests on the assumption that the senile plaque and Abeta peptide are causative of Alzheimer’s. A huge amount of money has been spent and lost on drugs (antibodies mostly) trying to get rid of the plaques. None have helped clinically. It is possible that the plaque is the last gasp of a neuron dying of something else (e.g. a tombstone rather than a smoking gun). It is also possible that the plaque is actually a way the neuron was defending itself against what was trying to kill it (e.g. the plaque as a pile of spent bullets).

Are you as smart as the (inanimate) blind watchmaker

Here’s a problem the cell has solved. Can you? Figure out a way to send a protein to two different membranes in the cell (the membrane encoding it { aka the plasma membrane }, and the endoplasmic reticulum) in the proportions you wish.

The proteins must have exactly the same sequence and content of amino acids, ruling out alternative splicing of exons in the mRNA (if this is Greek to you have a look at the following post — https://luysii.wordpress.com/2012/01/09/molecular-biology-survival-guide-for-chemists-v-the-ribosome/ and the others collected under — https://luysii.wordpress.com/category/molecular-biology-survival-guide/).

The following article tells you how the cell does it. Recall that not all of the messenger RNA (mRNA) is translated into protein. The ribosome latches on to the 5′ end of the mRNA,  subsequently moving toward the 3′ end until it finds the initiator codon (AUG which codes for methionine). This means that there is a 5′ untranslated region (5′ UTR). It then continues moving 3′ ward stitching amino acids together.  Similarly after the ribosome reaches the last codon (one of 3 stop codons) there is a 3′ untranslated region (3′ UTR) of the mRNA. The 3′ UTR isn’t left alone but is cleaved and a polyAdenine tail added to it. The 3′ UTR is where most microRNAs bind controlling mRNA stability (hence the amount of protein produced from a given mRNA).

The trick used by the cell is described in [ Nature vol. 522 pp. 363 – 367 ’15 ]. The 3’UTR is alternatively processed producing a variety of short and long 3’UTRs. One such protein where this happens is CD47 — which is found on the surface of most cells where it stops the cell from being eaten by scavenger cells such as macrophages. The long 3′ UTR of CD47 allows efficient cell surface expression, while the short 3′ UTR localizes it to the endoplasmic reticulum.

How could this possibly work? Once the protein is translated by the ribosome, it leaves the ribosome and the mRNA doesn’t it? Not quite.

They say that the long 3′ UTR of CD47 acts as a scaffold to recruit a protein complex which contains HuR (aka ELAVL1), an RNA binding protein and SET to the site of translation. The allows interaction of SET with the newly translated cytoplasmic domains of CD47, resulting in subsequent translocation of CD47 to the plasma membrane via activated RAC1.

The short 3′ UTR of CD47 doesn’t have the sequence binding HuR and SET, so CD47 doesn’t get to the plasma membrane, rather to the endoplasmic reticulum.

The mechanism may be quite general as HuR binds to thousands of mRNAs. The paper gives two more examples of proteins where this happens.

It’s also worth noting that all this exquisite control, does NOT involve covalent bond formation and breakage (e.g. not what we consider classic chemical reactions). Instead it’s the dance of one large molecular object binding to another in other ways. The classic chemist isn’t smiling. The physical chemist is.

Why drug discovery is so hard: Reason #26 — We’re discovering new players all the time

Drug discovery is so very hard because we don’t understand the way cells and organisms work very well. We know some of the actors — DNA, proteins, lipids, enzymes but new ones are being discovered all the time (even among categories known for decades such as microRNAs).

Briefly microRNAs bind to messenger RNAs usually decreasing their stability so less protein is made from them (translated) by the ribosome. It’s more complicated than that (see later), but that’s not bad for a first pass.

Presently some 2,800 human microRNAs have been annotated. Many of them are promiscuous binding more than one type of mRNA. However the following paper more than doubled their number, finding some 3,707 new ones [ Proc. Natl. Acad. Sci. vol. 112 pp. E1106 – E1115 ’15 ]. How did they do it?

Simplicity itself. They just looked at samples of ‘short’ RNA sequences from 13 different tissue types. MicroRNAs are all under 30 nucleotides long (although their precursors are not). The reason that so few microRNAs have been found in the past 20 years is that cross-species conservation has been used as a criterion to discover them. The authors abandoned the criterion. How did they know that this stuff just wasn’t transcriptional chaff? Two enzymes (DROSHA, DICER) are involved in microRNA formation from larger precursors, and inhibiting them decreased the abundance of the ‘new’ RNAs, implying that they’d been processed by the enzymes rather than just being runoff from the transcriptional machinery. Further evidence is that of half were found associated with a protein called Argonaute which applies the microRNA to the mRBNA. 92% of the microRNAs were found in 10 or more samples. An incredible 23 billion sequenced reads were performed to find them.

If that isn’t complex enough for you, consider that we now know that microRNAs bind mRNAs everywhere, not just in the 3′ untranslated region (3′ UTR) — introns, exons. MicroRNAs also bind pseudogenes, SINEes, circular RNAs, nonCoding RNAs. So it’s a giant salad bowl of various RNAs binding each other affecting their stability and other functions. This may be echoes of prehistoric life before DNA arrived on the scene.

It’s early times, and the authors estimate that we have some 25,000 microRNAs in our genome — more than the number of protein genes.

As always, the Category “Molecular Biology Survival Guide” found on the left should fill in any gaps you may have.

One rather frightening thought; If, as Dawkins said, we are just large organisms designed to allow DNA to reproduce itself, is all our DNA, proteins, lipids etc, just a large chemical apparatus to allow our RNA to reproduce itself? Perhaps the primitive RNA world from which we are all supposed to have arisen, never left.