Forgotten but not gone — take II

The RNA world from whence we sprang strikes again, this time giving us a glimpse into its own internal dynamic.  18 months ago I wrote the following post — which will give you the background to follow the latest (found at the end after the (***)

Life is said to have originated in the RNA world.  We all know about the big 3 important RNAs for the cell, mRNA, ribosomal RNA and transfer RNA.  But just like the water, sewer, power and subway systems under Manhattan, there is another world down there in the cell which doesn’t much get talked about.  These areRNAs, whose primary (and possibly only) function is to interact with other RNAs.

Start with microRNAs (of which we have at least 1,500 as of 12/12).  Their function is to bind to messenger RNA (mRNA) and inhibit translation of the mRNA into protein.  The effects aren’t huge, but they are a more subtle control of protein expression, than the degree of transcription of the gene.

Then there are ceRNAs (competitive endogenous RNAs) which have a large number of binding sites for microRNAs — humans have a variety of them all with horrible acronyms — HULC, PTCSC3 etc. etc. They act as sponges for microRNAs keeping them bound and quiet.

Then there are circular RNAs.  They’d been missed until recently, because typical RNA sequencing methods isolate only RNAs with characteristic tails, and a circular RNA doesn’t have any.  One such is called CiRS7/CDR1) which contain 70 binding sites for one particular microRNA (miR-7).  They are unlike to be trivial.  They are derived from 15% of actively transcribed genes.  They ‘can be’ 10 times as numerous as linear RNAs (like mRNA and everything else) — probably because they are hard to degrade < Science vol. 340 pp. 440 – 441 ’17 >. So some of them are certainly RNA sponges — but all of them?

The latest, and most interesting class are the nonCoding RNAs found in viruses. Some of them function to attack cellular microRNAs and help the virus survive. Herpesvirus saimiri a gamma-herpes virus establishes latency in the T lymphocytes of New World primates, by expressing 7 small nuclear uracil-rich nonCoding RNAs (called HSURs).  They associate with some microRNAs, and rather than blocking their function act as chaperones < Nature vol. 550 pp. 275 – 279 ’17 >.  They HSURs also bind to some mRNAs inhibiting their function — they do this by helping miR-16 bind to their targets — so they are chaperones.  So viral Sm-class RNAs may function as microRNA adaptors.

Do you think for one minute, that the cell isn’t doing something like this.

I have a tendency to think of RNAs as always binding to other RNAs by classic Watson Crick base pairing — this is wrong as a look at any transfer RNA structure will show. https://en.wikipedia.org/wiki/Transfer_RNA.  Far more complicated structures may be involved, but we’ve barely started to look.

Then there are the pseudogenes, which may also have a function, which is to be transcribed and sop up microRNAs and other things — I’ve already written about this — https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/.  Breast cancer cells think one (PTEN1) is important enough to stop it from being transcribed, even though it can’t be translated into protein.

*****

[ Proc. Natl. Acad. Sci. vol. 116 pp. 7455 – 7464 ’19 ] The work reports a fascinating example of that early world in which the function of one denizen (a circular RNA called cPWWP2A) binds to another denizen of that world (microRNA 579 aka miR-579) acting as a sponge sopping up so it can’t bind to the mRNAs for angiopoetitin1, occludin and SIRT1.

So what you say?  Well it may lead to a way to treat diabetic retinopathy. How did they find cPWWP2A?  They used the Shanghai BIotechnology Company Mouse Circular RNA microArray which measures circular RNAs.  They found that 400 or so that were upregulated in diabetic retinopathy and another 400 or so that were downregulated.  cPWWP2A was on of the 3 top upregulated circular RNAs in diabetic retinopathy.  cPWWP2A comes from (what else?) PWWP2A, a gene coding for a protein which specifically binds the histone protein H2A.Z.

Overexpression of cPWW2PA or inhibition of miR-579 improves retinal vascular dysfunction in experimental diabetes.

So here is all this stuff going on way down there in the RNA world, first interacting with other players in this world and eventually reaching up to the level we thought we knew about and controlling gene expression.  It’s sort of like DOS (Disc Operating System) still being important in Windows.

How much more stuff like this is to be discovered controlling gene expression in us is anyone’s guess

Why drug discovery is hard #29 — a very old player doing a very new thing

We all know what RNA does don’t we?  It binds to other RNAs and to DNA.  Sure lots of new forms of RNA have been found: microRNAs, competitive endogenous RNA (ceRNA), long nonCoding (for protein) RNA (lncRNA), piwiRNAs, small interfering RNAs (siRNAs), . .. The list appears endless.  But the basic mechanism of action of RNA in the cell is binding to some other polynucleotide (RNA or DNA) and affecting its function.

Not so fast.  A new paper http://science.sciencemag.org/content/358/6366/1051 describes  lncRNA-ACOD1, a cellular RNA induced by a variety of viruses.  lncRNA-ACOD1 binds to an enzyme enhancing its catalytic efficiency.  Now that’s new.  Certainly RNAs and proteins bind to each other in the ribosome, and in RNAase P, but here the proteins serve to structure the RNA so it can carry out its catalytic function, not the other way around.

The enzyme bound is called GOT2 (Glutamic Oxaloacetic Transaminase 2).  Much interesting cellular biochemistry is discussed in the paper which I’ll skip, except to say that the virus uses the hyped up GOT2 to repurpose the cell’s metabolic machinery for its own evil ends.

lncRNA-ACOD1 has 3 exons and a polyAdenine tail.  There are two transcript variants containing  2,330 and 2,259 nucleotides.  There are only 100 copies/cell.  lncRNA-ACOD1 nucleotides #165 – #390 bind to amino acids #54 – #68 of GOT2.

So what are the other 2000 or so nucleotides of lncRNA-ACOD1 doing?   The phenomenon of RNA binding to protein is quite likely to be more widespread.  Both the GOT2 interacting motif and the interacting sequence of lncRNA-ACOD1 are well conserved across species of hosts and viruses.

Although viruses co-opt lncRNA-ACOD1, it is normally expressed in the heart as is GOT2 with no viral infection at all.  So we have likely stumbled onto an entirely new method of cellular metabolic control, AND a whole new set of players and interactions for drugs to act on (if they aren’t already doing this unknown to us).

This is series member #29 of why drug development is hard, most of which concentrated on the fact that we don’t know all the players.  lncRNA-ACOD1 is different — RNA is a player we’ve known for a very long time  but it appears to be playing a game entirely new to us.

It is also good to see cutting edge research like this coming out of China.  Hopefully it will stand up, but enough questionable stuff has come from them that every Chinese paper is under a cloud.

This is why I love reading the current literature.  You never know what you’re going to find.  It’s like opening presents.

Forgotten but not gone

Life is said to have originated in the RNA world.  We all know about the big 3 important RNAs for the cell, mRNA, ribosomal RNA and transfer RNA.  But just like the water, sewer, power and subway systems under Manhattan, there is another world down there in the cell which doesn’t much get talked about.  These are RNAs, whose primary (and possibly only) function is to interact with other RNAs.

Start with microRNAs (of which we have at least 1,500 as of 12/12).  Their function is to bind to messenger RNA (mRNA) and inhibit translation of the mRNA into protein.  The effects aren’t huge, but they are a more subtle control of protein expression, than the degree of transcription of the gene.

Then there are ceRNAs (competitive endogenous RNAs) which have a large number of binding sites for microRNAs — humans have a variety of them all with horrible acronyms — HULC, PTCSC3 etc. etc. They act as sponges for microRNAs keeping them bound and quiet.

Then there are circular RNAs.  They’d been missed until recently, because typical RNA sequencing methods isolate only RNAs with characteristic tails, and a circular RNA doesn’t have any.  One such is called CiRS7/CDR1) which contain 70 binding sites for one particular microRNA (miR-7).  They are unlike to be trivial.  They are derived from 15% of actively transcribed genes.  They ‘can be’ 10 times as numerous as linear RNAs (like mRNA and everything else) — probably because they are hard to degrade < Science vol. 340 pp. 440 – 441 ’17 >. So some of them are certainly RNA sponges — but all of them?

The latest, and most interesting class are the nonCoding RNAs found in viruses. Some of them function to attack cellular microRNAs and help the virus survive. Herpesvirus saimiri a gamma-herpes virus establishes latency in the T lymphocytes of New World primates, by expressing 7 small nuclear uracil-rich nonCoding RNAs (called HSURs).  They associate with some microRNAs, and rather than blocking their function act as chaperones < Nature vol. 550 pp. 275 – 279 ’17 >.  They HSURs also bind to some mRNAs inhibiting their function — they do this by helping miR-16 bind to their targets — so they are chaperones.  So viral Sm-class RNAs may function as microRNA adaptors.

Do you think for one minute, that the cell isn’t doing something like this.

I have a tendency to think of RNAs as always binding to other RNAs by classic Watson Crick base pairing — this is wrong as a look at any transfer RNA structure will show. https://en.wikipedia.org/wiki/Transfer_RNA.  Far more complicated structures may be involved, but we’ve barely started to look.

Then there are the pseudogenes, which may also have a function, which is to be transcribed and sop up microRNAs and other things — I’ve already written about this — https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/.  Breast cancer cells think one (PTEN1) is important enough to stop it from being transcribed, even though it can’t be translated into protein.

Why our brains are large: the elegance of its molecular biology

Primates have much larger brains in proportion to their body size than other mammals. Here’s why. The mechanism is incredibly elegant. Unfortunately, you must put a sizable chunk of recent molecular biology under your belt before you can comprehend it. Anyone can listen to Mozart without knowing how to read or write music. Not so here.

I doubt that anyone can start from ground zero and climb all the way up, but here is all the background you need to comprehend what follows. Start here — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
and follow the links (there are 5 more articles).

Also you should be conversant with competitive endogenous RNA (ceRNA) — here’s a link — https://luysii.wordpress.com/2014/01/20/why-drug-discovery-is-so-hard-reason-24-is-the-3-untranslated-region-of-every-protein-a-cerna/

Also you should understand what microRNAs are — we’re still discovering all the things they do — here’s the background you need — https://luysii.wordpress.com/2015/03/22/why-drug-discovery-is-so-hard-reason-26-were-discovering-new-players-all-the-time/weith.

Still game?

Now we must delve into the embryology of the brain, something few chemists or nonbiological type scientists have dealt with.

You’ve probably heard of the term ‘water on the brain’. This refers to enlargement of the ventricular system, a series of cavities in all our brains. In the fetus, all nearly all our neurons are formed from cells called neuronal precursor cells (NPCs) lining the fetal ventricle. Once formed they migrate to their final positions.

Each NPC has two choices — Choice #1 –divide into two NPCs, or Choice #2 — divide into an NPC and a daughter cell which will divide no further, but which will mature, migrate and become an adult neuron. So to get a big brain make NPCs adopt choice #1.

This is essentially a choice between proliferation and maturation. It doesn’t take many doublings of a NPC to eventually make a lot of neurons. Naturally cancer biologists are very interested in the mechanism of this choice.

Well to make a long story short, there is a protein called NOTCH — vitally important in embryology and in cancer biology which, when present, causes NPCs to make choice #1. So to make a big brain keep Notch around.

Well we know that some microRNAs bind to the mRNA for NOTCH which helps speed its degradation, meaning less NOTCH protein. One such microRNA is called miR-143-3p.

We also know that the brain contains a lncRNA called lncND (ND for Neural Development). The incredible elegance is that there is a primate specific insert in lncND which contains 16 (yes 16) binding sites for miR-143-3p. So lncND acts as a sponge for miR-143-3p meaning it can’t bind to the mRNA for NOTCH, meaning that there is more NOTCH around. Is this elegant or what. Let’s hear it for the Blind Watchmaker, assuming you have the faith to believe in such things.

Fortunately lncND is confined to the brain, otherwise we’d all be dead of cancer.

Should you want to read about this, here’s the reference [ Neuron vol. 90 pp. 1141 – 1143, 1255 – 1262 ’16 ] where there’s a lot more.

Historically, this was one of the criticisms of the Star Wars Missile Defense — the Russians wouldn’t send over a few missles, they’d send hundreds which would act as sponges to our defense. Whether or not attempting to put Star Wars in place led to Russia’s demise is debatable, but a society where it was a crime to own a copying machine, could never compete technically to produce such a thing.

SmORFs and DWORFs — has molecular biology lost its mind?

There’s Plenty of Room at The Bottom is a famous talk given by Richard Feynman 56 years ago. He was talking about something not invented until decades later — nanotechnology. He didn’t know that the same advice now applies to molecular biology. The talk itself is well worth reading — here’s the link http://www.zyvex.com/nanotech/feynman.html.

Those not up to speed on molecular biology can find what they need at — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/. Just follow the links (there are only 5) in the series.

lncRNA stands for long nonCoding RNA — nonCoding for protein that is. Long is taken to mean over 200 nucleotides. There is considerable debate concerning how many there are — but “most estimates place the number in the tens of thousands” [ Cell vol. 164 p. 69 ’16 ]. Whether they have any cellular function is also under debate. Could they be like the turnings from a lathe, produced by the various RNA polymerases we have (3 actually) simply transcribing the genome compulsively? I doubt this, because transcription takes energy and cells are a lot of things but wasteful isn’t one of them.

Where does Feynmann come in? Because at least one lncRNA codes for a very small protein using a Small Open Reading Frame (SMORF) to do so. The protein in question is called DWORF (for DWorf Open Reading Frame). It contains only 34 amino acids. Its function is definitely not trivial. It binds to something called SERCA, which is a large enzyme in the sarcoplasmic reticulum of muscle which allows muscle to relax after contracting. Muscle contraction occurs when calcium is released from the endoplasmic reticulum of muscle.  SERCA takes the released calcium back into the endoplasmic reticulum allowing muscle to contract. So repetitive muscle contraction depends on the flow and ebb of calcium tides in the cell. Amazingly there are 3 other small proteins which also bind to SERCA modifying its function. Their names are phospholamban (no kidding) sarcolipin and myoregulin — also small proteins of 52, 31 and 46 amino acids.

So here is a lncRNA making an oxymoron of its name by actually coding for a protein. So DWORF is small, but so are its 3 exons, one of which is only 4 amino acids long. Imagine the gigantic spliceosome which has a mass over 1,300,000 Daltons, 10,574 amino acids making up 37 proteins, along with several catalytic RNAs, being that precise and operating on something that small.

So there’s a whole other world down there which we’ve just begun to investigate. It’s probably a vestige of the RNA world from which life is thought to have sprung.

Then there are the small molecules of intermediary metabolism. Undoubtedly some of them are used for control as well as metabolism. I’ll discuss this later, but the Human Metabolome DataBase (HMDB) has 42,000 entries and METLIN, a metabolic database has 240,000 entries.

Then there is competitive endogenous RNA –https://luysii.wordpress.com/2012/01/29/why-drug-discovery-is-so-hard-reason-20-competitive-endogenous-rnas/

Do you need chemistry to understand this? Yes and no. How the molecules do what they do is the province of chemistry. The description of their function doesn’t require chemistry at all. As David Hilbert said about axiomatizing geometry, you don’t need points, straight lines and planes You could use tables, chairs and beer mugs. What is important are the relations between them. Ditto for the chemical entities making us up.

I wouldn’t like that.  It’s neat to picture in my mind our various molecular machines, nuts and bolts doing what they do.  It’s a much richer experience.  Not having the background is being chemical blind..  Not a good thing, but better than nothing.

The old year goes out with a bang

A huge amount of cellular genomics will have to be redone if the following paper is replicated. Remember “Extraordinary claims require extraordinary evidence.” Carl Sagan.

What’s all the shouting about? Normally when you think about messenger RNA (mRNA) as it exists in the cytoplasm after the initial transcript is significantly massaged in the nucleus, you think about the part that codes for amino acids. This ‘coding region’ is the part that is translated into amino acids by the ribosome. But mRNA is invariably larger having nucleotides at each end (3′ and 5′) which have other uses. These are called the 3′ Untranslated Region (3′ UTR) and 5′ Untranslated Region (5′ UTR).

So if you do single cell RNA sequencing (which we can do now) it shouldn’t matter what nucleotide sequence you search for (5′ UTR, 3′ UTR or the coding region) as all mRNA contains one of each.

Not so says this paper [ Neuron vol. 88 pp. 1149 – 1156 ’15 ].

Given the mRNA for a given protein in a single cell, using a probe for the 3’UTR and a probe for the coding sequence should give you the same abundance for both. That’s not what they found at all for single neurons from the brain. In some cases there was much more RNA coding for the 3’UTR than for the coding segment of a given mRNA for a protein. In others there was much less. Even more impressively is that the 3’UTR/(3’UTR + coding) ratio for a given protein varies between different parts of the brain. Obviously this ratio should be .5 given what we knew about mRNA in the past. The ratio has to be between 0 and 1.

Well they looked at a lot of proteins. The did find around 1,400 genes with a ratio of .5 (as expected), but they found 700 showing a ratio of .2 (lots more 3’UTR than coding sequence), and 1,100 showing a ratio of .8. Overall plotting the ratio vs. number of genes with that ratio gives something looking like a bell curve (Gaussian distribution).

It’s long been known that mRNA levels don’t exactly correlate with the levels of proteins made from them. If there’s lots of 3’UTRs around the authors found that there was relatively little protein made from the gene.

A variety of brain atlases have published mRNA abundances for various regions of the brain. If they just used one probe (as they probably did) this is clearly not enough.

The 3’UTRs may be acting as ceRNAs (competitive endogenous RNAs). These have been known for years — I’ve included a post of 3 years ago on the subject (at the end).

So this work (if replicated) throws everything we thought we knew about mRNA into a cocked hat. It’s why I love science, there’s always something really new to think about. Happy New Year !!!

Chemiotics II
Lotsa stuff, basically scientific — molecular biology, organic chemistry, medicine (neurology), math — and music
Why drug discovery is so hard: reason #20 — competitive endogenous RNAs

The chemist will appreciate le Chatelier’s principle in action in what follows. We are far from knowing all the players controlling cellular behavior. So how in the world will we find drugs to change cellular behavior when we don’t know all the things affecting it. The latest previously unknown cellular player to enter the lists are competitive endogenous RNAs (ceRNAs). For details see Cell vol. 147 pp. 344 – 357, 382 – 395 ’11. The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

Recall that microRNAs are short (20 something) polynucleotides which bind to the 3′ untranslated region (3′ UTR) of mRNA, and either (1) inhibit its translation into protein (2) cause its degradation. In each case, less of the corresponding protein is made. The microRNA and the appropriate sequence in the 3′ UTR of the mRNA form an RNA-RNA double helix (G on one strand binding to C on the other, etc.). Visualizing such helices is duck soup for a chemist.

Molecular biology is full of such semantic cherry bombs as nonCoding DNA (which meant DNA which didn’t cord for protein), a subset of Junk DNA. Another is the pseudogene — these are genes that look like they should code for protein, except that they don’t because of lack of an initiation codon or a premature termination codon. Except for these differences, they have the nucleotide sequence to code for a known protein. It is estimated that the human genome contains as many pseudogenes (20,000) as it contains true protein coding genes [ Genome Res. vol. 12 pp. 272 – 280 ’02 ]. We now know that well over half the genome is transcribed into mRNA, including the pseudogenes.

PTEN (you don’t want to know what it stands for) is a 403 amino acid protein which is one of the most commonly mutated proteins in human cancer. Our genome also contains a pseudogene for it (called PTENP). Interestingly deletion of PTENP (not PTEN) is found in some cancers. However PTENP deletion is associated with decreased amounts of the PTEN protein itself, something you don’t want as PTEN is a tumor suppressor. How PTEN accomplishes this appears to be fairly well known, but is irrelevant here.

Why should loss of PTENP decrease PTEN itself? The reason is because the mRNA made from PTENP, even though it has a premature termination codon, and can’t be made into protein, is just as long, so it also contains the 3’UTR of PTEN. This means PTENP is sopping up microRNAs which would otherwise decrease the level of PTEN. Think of PTENP mRNA as a sponge.

Subtle isn’t it? But there’s far more. At least PTENP mRNA closely resembles the PTEN mRNA. However other mRNAs coding for completely different proteins, also have binding sites in their 3’UTR for the microRNA which binds to the 3UTR of PTEN, resulting in its destruction. So transcription of a completely different gene (the example of ZEB2 is given) can control the abundance of another protein. Essentially its mRNA is acting as a sponge, sopping up the killer microRNA.

It gets worse. Most microRNAs have binding sites on the mRNAs of many different proteins, and PTEN itself has a 3’UTR which binds to 10 different microRNAs.

So here is a completely unexpected mechanism of control of protein levels in the cell. The general term for this is competitive endogenous RNA (ceRNA). Two years ago the number of human microRNAs was thought to be around 1,000. Unlike protein coding genes, it’s far from obvious how to find them by looking at the sequence of our genome, so there may be quite a few more.

So most microRNAs bind the 3’UTR of more than one protein (the average number is unclear at this point), and most proteins have binding sites for microRNAs in their 3’UTR (again the average number is unclear). What a mess. What subtlety. What an opportunity for the regulation of cellular function. Who is going to be smart enough to figure out a drug which will change this in a way that we want. Absence of evidence of a regulatory mechanism is not evidence of its absence. A little humility is in order.

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.

Why drug discovery is so hard: Reason #24 — Is the 3′ untranslated region of every mRNA a ceRNA?

We all know what proteins do. They act as enzymes, structural elements of cells, membrane proteins where drugs bind etc. etc. The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

We also know that that the messenger RNA for any given protein contains a lot more information than that needed to code for the amino acids making up the protein. Forget the introns that are spliced out from the initial transcript. When the mature messenger RNA for a given protein leaves the nucleus for the cytoplasm where the ribosome translates it into protein at either end it contains nucleotides which the ribosome effectively ignores. These are called the untranslated regions (UTRs). The UTRs at the 3′ end of human mRNAs range in length between 60 and 4,000 nucleotides (average 800). It costs energy to store the information for the UTR in DNA, more energy to synthesize the nucleotides which make it up, even more to patch them together to form the UTR, more to package it and move it out of the nucleus etc. etc.

Why bother? Because the 3′ UTR of the mRNA contains a lot of information which tells the cell how much protein to make, how long the mRNA should hang around in the cell (among many other things). A Greek philosopher got here first — “Nature does nothing uselessly” – Aristotle

Those familiar with competitive endogenous RNA (ceRNA) can skip what follows up to the ****

Recall that microRNAs are short (20 something) polynucleotides which bind to the 3′ untranslated region (3′ UTR) of mRNA, and either (1) inhibit its translation into protein (2) cause its degradation. In each case, less of the corresponding protein is made. The microRNA and the appropriate sequence in the 3′ UTR of the mRNA form an RNA-RNA double helix (G on one strand binding to C on the other, etc.). Visualizing such helices is duck soup for a chemist.

Molecular biology is full of such semantic cherry bombs as nonCoding DNA (which meant DNA which didn’t cord for protein), a subset of Junk DNA. Another is the pseudogene — these are genes that look like they should code for protein, except that they don’t because of lack of an initiation codon or a premature termination codon. Except for these differences, they have the nucleotide sequence to code for a known protein. It is estimated that the human genome contains as many pseudogenes (20,000) as it contains true protein coding genes [ Genome Res. vol. 12 pp. 272 – 280 ’02 ]. We now know that well over half the genome is transcribed into mRNA, including the pseudogenes.

PTEN (you don’t want to know what it stands for) is a 403 amino acid protein which is one of the most commonly mutated proteins in human cancer. Our genome also contains a pseudogene for it (called PTENP). Interestingly deletion of PTENP (not PTEN) is found in some cancers. However PTENP deletion is associated with decreased amounts of the PTEN protein itself, something you don’t want as PTEN is a tumor suppressor. How PTEN accomplishes this appears to be fairly well known, but is irrelevant here.

Why should loss of PTENP decrease PTEN itself? The reason is because the mRNA made from PTENP, even though it has a premature termination codon, and can’t be made into protein, is just as long, so it also contains the 3′UTR of PTEN. This means PTENP is sopping up microRNAs which would otherwise decrease the level of PTEN. Think of PTENP mRNA as a sponge.

Subtle isn’t it? But there’s far more. At least PTENP mRNA closely resembles the PTEN mRNA. However other mRNAs coding for completely different proteins, also have binding sites in their 3′UTR for the microRNA which binds to the 3UTR of PTEN, resulting in its destruction. So transcription of a completely different gene (the example of ZEB2 is given) can control the abundance of another protein. Essentially its mRNA is acting as a sponge, sopping up the killer microRNA.

It gets worse. Most microRNAs have binding sites on the mRNAs of many different proteins, and PTEN itself has a 3′UTR which binds to 10 different microRNAs.

So here is a completely unexpected mechanism of control of protein levels in the cell. The general term for this is competitive endogenous RNA (ceRNA). Two years ago the number of human microRNAs was thought to be around 1,000 (release 2.0 of miRBase in June ’13 gives the number at 2,555 — this is unlikely to be complete). Unlike protein coding genes, it’s far from obvious how to find them by looking at the sequence of our genome, so there may be quite a few more.

So most microRNAs bind the 3′UTR of more than one protein (the average number is unclear at this point), and most proteins have binding sites for microRNAs in their 3′UTR (again the average number is unclear). What a mess. What subtlety. What an opportunity for the regulation of cellular function. Who is going to be smart enough to figure out a drug which will change this in a way that we want. Absence of evidence of a regulatory mechanism is not evidence of its absence. A little humility is in order.

*****

If this wasn’t a scary enough, consider the following cautionary tale — Nature vol. 505 pp. 212 – 217 ’14. HMGA2 is a protein we thought we understood for the most part. It is found in the nucleus, where it binds to DNA. While it doesn’t transcribe DNA into RNA, it does bind to DNA helping to form a protein complex which binds to DNA which effectively helps promote transcription of certain genes.

Well that’s what the protein does. However the mRNA for the protein uses its 3′ untranslated region (3’UTR) to sop up microRNAs of the let-7 family. The mRNA for HMGA2 is highly overexpressed in human cancer (notably the very common adenocarcinoma of the lung). You can mutate the mRNA for HMGA2 so it doesn’t produce the protein, just by putting a stop codon in it near the 5′ end. Throw the altered mRNA into a tissue culture of an lung adenocarcinoma cell line, and the cell become more proliferative and grows independently of being anchored to the tissue culture plate (e.g. anchorage independence, a biologic marker for cancer).

So what? It means that it is possible that every mRNA for every protein we make is acting as a ceRN A. The authors conclude the paper with ” Such dual-function ceRNA and protein activities necessitate a deeper exploration of the coding genome in biological systems.”

I’ll say. We’re just beginning to scratch the surface. The control mechanisms within the cell continue to amaze (me) by their elegance and subtlety. I doubt highly that we know them all. Yet more reasons that drug discovery is hard — we are mucking about with a system whose workings we only dimly understand.