Back to the drawing board on knockouts and knockdowns

Nothing could be simpler than the distinction between the initial product of genes that code for proteins (mRNA) and genes that don’t (long non-Coding RNAs — aka lncRNA, lincRNA). Not anymore according to an exceedingly clever and well thought out piece of work.

[ Cell vol. 168 pp. 753 – 755, 843 – 855 ’17 ] We know that ultraviolet light damages DNA primarily by forming pyrimidine dimers. Naturally transcription of DNA won’t be as accurate, so the cell has ways to shut it down. Ultraviolet exposure results in an unusual type of restriction of transcription along with slower elongation, with the result that only the promoter proximal 20 – 25 kiloBases of a protein coding gene are efficiently transcribed into mRNA.

In addition, after ultraviolet damage there is a global switch in pre-mRNA processing resulting in a preference for the production of transcripts containing alternative last exons not normally included in the dominant mRNA isoform. Some 84 genes are processed this way.

ASCC3 is the strongest regulator of transcription following UV damage, acting to repress it after UV damage. It is a DEAD/DEAH box DNA helicase component. The ASCC3 protein interacts with RNA polymerase II (Pol II) and becomes highly ubiquitinated and phosphorylated on UV irradiation. It isn’t required to establish transcriptional repression, just maintainance. Disruption of the UV specific form — e.g. the short isoform containing the alternative last exon has the opposite effect, allowing transcriptional recovery after UV damage.

This explains why the human genes remaining expressed (or actually induced) after UV irradiation are invariably ‘very short’ (whatever that means).

The short and long isoforms constitute an autonomous regulatory module, and are related functionally, so the effect of deleting one can at least be partially compensated for by deleting the other.

The 3,100 nucleotide long ‘short’ isoform, codes for a protein, but the protein itself didn’t have the effect of the short form mRNA (see if you can figure out, without reading further how the authors proved this). The mRNA produced from the short isoform is found almost exclusively in the nucleus. The authors put in a stop codon immediately downstream of the start codon which ablated protein production but not transcription into the appropriate mRNA, but there was still rescue of the transcriptional recovery phenotype. So the functional form of the short RNA isoform is mediated by a nonCoding RNA encoded in the ASCC3 protein coding gene. The short ASCC3 isoform has an open reading frame of 333 nucleotides, but functionally it is a lncRNA (of 3.5 kiloBases).

So protein genes can produce functional lncRNAs. How many of them actually do this is unknown. When you knockdown a gene, how much of the effect is due to less protein and how much due to the (putative) lncRNA which also might be produced by the gene. That’s why it’s back to the drawing board for knockout mice (or even mRNA knockdown using shRNA etc. etc.)

The current definition of lncRNA is absence of protein coding potential in a gene.

Why have the same gene code for two different things — there may be a regulatory advantage — controlling the function of the protein. lncRNAs have the unique ability to act in close spatial proximity to their transcription loci.

Stay tuned. It’s just fascinating what we still don’t know.

An incredible paper which may turn diabetes treatment on its head

First off, why should a neurologist be interested in diabetes?  That’s for endocrinologists isn’t it?  Not really.  One of the most serious aspects of diabetes is its acceleration of vascular disease (atherosclerosis), with an increased risk of heart attack and stroke (which is where the neurologist comes in).  So you have a diabetic who has survived a stroke. You want to prevent the next one, and you know that they’re in a much higher risk group for another stroke (1) because they just had one (2) because they’re more likely to have another because of their diabetes.  Also vascular disease makes any neurologic problem worse, dementia, neuropathy you name it.

So I’ve always tried to stay current on diabetes.  Which brings me to Proc. Natl. Acad. Sci. vol. 109 pp. 14972 – 14976 ’12 (11 September issue).  You’d think that nearly a century after Banting and Best’s discovery of Insulin that there were no new wrinkles left.  Not so.

Now for a bit of anatomy and physiology.  In a very simplistic way, you can regard diabetes as not enough insulin.  Insulin lowers blood sugar and is needed after you eat.   It is made in the pancreas, an organ which secretes digestive enzymes into your gastrointestinal tract.  It also secretes insulin, but into the blood rather than the gut.  Insulin is made in relatively small collections of cells (1,000 – 10,0000) called islets dispersed through out the pancreas.  They are .1 – .2 milliMeters in diameter, and even though we have over 1,000,000 of them, they constitute 2% of the mass of the pancreas. 85% of the islet cells make insulin (the beta cells), another cell type (the alpha cells) make another protein (glucagon), also secreted into the blood, making it a hormone by definition.  Glucagon raises blood sugar, by causing the liver to make glucose and then pump it out.

Streptozotocin (a glucose derivative) made by soil bacteria, has the nasty property of selectively killing pancreatic beta cells, leaving everything else alone.  Naturally experimentalists love it and have used it to produce severe diabetes (fatal if untreated) in lab animals, and then try new treatments to see if they can come up with something better.

Amazingly, here’s one they didn’t try until now.  Given our present tools, you can pretty much knock out any gene you want in a mouse embryonic stem cell, implant it in another mouse, and (after a lot of failure), make a strain of mice lacking that particular gene (these are what’s called knockout mice).  What they knocked out was not glucagon (which has effects all over the body, including the brain), but the receptor for glucagon.  So even though there’s plenty of glucagon around, lacking the receptor cells don’t respond to it.  It’s like hearing a language you don’t know — the sound is there all right but you can’t internalize it and react to what’s being said.

It had been known for a while that giving streptozotocin to mice lacking the glucagon receptor doesn’t produce diabetes (or much else).  The finding as been treated with a good deal of skepticism, being blamed on other factors.  The authors of the present paper, found a way to put the glucagon receptor back into the liver (using a virus).  When they did this to a knockout mouse living happily despite being treated with strpetozotocin, their glucose shot up.  The virus didn’t hang around forever, glucagon receptor levels in the liver dropped and blood sugar dropped along with it.   Remember that a normal mouse dies of diabetic complications fairly quickly after receiving streptozotocin.

You couldn’t ask for much better proof, and a new way to treat diabetes may have been found.  Amazing.

As in all of medicine there are caveats.  The glucagon receptor is found in other organs, heart and brain among them, so blocking the action of glucagon this way  may have many other effects.

We wouldn’t exist if retroviruses weren’t moving around in our genome.

Time for some of the excellent molecular biology I’ve put off writing about while I plow through the new Clayden.  I reached the halfway point today (p. 590) Exactly 2 months and 2 weeks after it arrived.  The chemist might need  some brushing up on DNA and messenger RNA before pushing on.  Pretty much all the background needed is found in https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ an d https://luysii.wordpress.com/2010/07/11/molecular-biology-survival-guide-for-chemists-ii-what-dna-is-transcribed-into/.

Everyone has heard of the AIDs virus.  It has so far been impossible to cure because it hides in our DNA doing next to nothing.  Tickle it in a variety of unknown ways, and it’s DNA is transcribed into messenger RNA (mRNA), the virus is assembled and goes on to wreak havoc with our immune system.  How does the AIDs virus get into our DNA in the first place?  Its genome is made of RNA, not DNA.  It has an enzyme (reverse transcriptase) which transcribes its RNA into DNA, and another enzyme (the integrate, which is actually a complex of proteins) which patches the DNA copy (called cDNA) into our genome.  That’s why we can’t get rid of it.  That’s also why it’s called a retrovirus — because of retrograde transcription of its RNA into cDNA).

Well, sorry to say, but at least 10% of our DNA is made of retrovirus remnants.  The vast majority of them have been crippled by mutation so their reverse transcriptases  don’t work any more, or there is something wrong with their integrase, etc. etc.  Some of them do make RNA copies of themselves however, but the copies are mutated enough that infectious virus doesn’t form.  But the RNA copies can be reverse transcribed  into cDNA and reinserted back into our DNA, and in a new site to boot.  This is why they are called retrotransposons.

The whole bunch of retroviruses, retrotransposons, and other repetitive elements of DNA have been called ‘junk’ by eminent authority.  Another epithet for them is the selfish gene — which exists only to reproduce itself.  Humans are said to be machines for reproducing human DNA.

Enter  [ Cell vol. 150 pp. 7 – 9, 29 – 38 ’12 ].  Now it’s time for some very human biology The fetus represents an immunologically different graft to the mother.  Half its antigens are tolerated because they are maternal, the paternal half are not likely to be.  Allogeneic means a transplant from a different member of the same species, so the fetus is regarded as semiallogeneic. 

So why doesn’t our immune system attack the placenta surrounding the fetus, which expresses the paternal proteins?  There’s probably a lot more to it but a class of immune cell called a regulatory T cell (Treg) shuts down the immune response wherever they are found, and the placenta has lots of them.

Different cells express different proteins, and Tregs are no exception. A transcription factor is something that binds to the DNA in front of a gene, turning on transcription of the gene,  ultimately increasing production of the protein the gene codes for. Specificity is obtained by the transcription factor binding to particular sequences of DNA, which are found in only in front of a subset of  genes

The transcription factor which turns on genes necessary to turn an immune cell into a Treg is called Foxp3.  Foxp3 is a protein and to have lots of it around the gene for it must be turned on so its mRNA can be made.  Guess what?  This means that other transcription factors must bind in front the Foxp3 gene.

Here’s Jonathan Swift on the subject

So nat’ralists observe, a flea

Hath smaller fleas that on him prey,

And these have smaller fleas that bite ’em,

And so proceed ad infinitum.”

…

An important protein like Foxp3 is highly controlled.  There are 3 distinct regions in front of the gene were other transcription factors and repressors of transcription bind.  They are called conserved nonCoding sequences (CNSs), an oxymoron, because they are clearly coding for something quite important. The 3 sequences are called CNS1, CNS2 and CNS3.    Technology has progressed to the point where we can remove just about any DNA sequence from the mouse genome we wish (the resultant mice are called knockout mice).  

Anyway if you knockout CNS1 the mice resorb semiallogenic fetuses (where the father and the mother aren’t genetically related), but not allogenic fetuses (where the genomes of the father and the mother are pretty much the same due to inbreeding).  It’s possible to trace Foxp3 far back in evolution.  Only animals with placentas (eutherians) have CNS1 in addition to CNS2 and CNS3. Marsupials, which don’t have placentas, just have CNS2 and CNS3. 

So where do retrotransposons come in?  The structure of CNS1 shows that it is a retrotransposon which moved in front of the Foxp3 gene.  It mutated enough for a new and different set of transcription factors to bind to it and turn on Foxp3 expression in the placenta allowing survival of the fetus.  Some Junk DNA indeed !