Forgotten but not gone — take III

It’s pretty clear that life originated in the RNA world.  Consumed by thinking of proteins, enzymes, DNA etc. we tend to forget that there is a lot of RNA out there doing things we didn’t suspect.  Here are two more examples, one of which may explain why even genes coding  for proteins are relatively free of codons transcribed into amino acids.  The champ of course is dystrophin, discussed in the last post — https://luysii.wordpress.com/2019/05/05/duchenne-muscular-dystrophy-a-novel-genetic-treatment/.  The gene is a monster with  2,220,233 nucleotides coding for just 3,685 amino acids, meaning that less than 1/200th of the gene is actually coding for protein. The work below should make us think about just what else the 199/200th of dystrophin might be doing,

Unsuspected use of RNA #1.   [ Neuron vol. 102 pp. 507 – 509, 553 – 563 ’19 ]  The Tumor protein p53 inducible nuclear protein 2 (Tp53inp2) gene codes for a low complexity protein of 222 amino acids, all in one exon.  However the ‘3 untranslated region (3’UTR)  of the RNA for it is nearly 5 times longer (3,121 nucleotides) vs. 666 amino acid coding nucleotides.  The protein is made from the mRNA in some cells, but not in sympathetic neurons, even though the mRNA for Tp53inp2 is the most abundant RNA in the axons of these neurons.

Why do animals lick their wounds?  Because their saliva contains nerve growth factor (NGF) among other things.  NGF is crucial for the growth of sympathetic neuron axons, and their very survival in embryonic life.  It is a protein, which binds to a receptor for it (TrkA) on the axon membrane.  The receptor/NGF complex is then internalized and transported back to the nucleus turning on the genes necessary for axon growth and cell survival.

Even though the mRNA for Tp53inp2 is NOT translated into protein in the axon, it is crucial for the internalization of TrkA/NGF.

People have studied proteins whose function it is to bind RNA for years.  They are called RBPs (RNA Binding Proteins), and our genome has 750 of them.  200 RBPs are associated with genetic disease.  This work turns everthing on its head.  Here is an RNA whose function it is to bind a protein (e.g. TrkA).

How many more mRNAs have nonCoding (for protein) parts with other functions?

Unsuspected use of RNA #2. Circular RNAs had been missed for years (although known since 1976).  The classic sequencing methods isolate only RNAs with characteristic tails (such as polyAdenine).  Circular RNAs don’t have any.    They are formed by back splicing of 3′ end of exon N to the 5′ end of exon N.  Fortunately this is only 1% as efficient as the normal way.

So what?  Circular RNAs are crucial in the innate immune response to microbial invaders.  Double stranded DNA belongs inside the nucleus.  When it gets into the cytoplasm when some organism brings it there,it binds to Protein Kinase R (PKR) activating it so it phosphorylates eukaryotic initiation factor 2 (eiF2) bringing protein synthesis to a screeching halt.

This means that the cell needs a mechanism to keep PKR quiet.  This is where circular RNAs come in   [ Cell vol. 177 pp. 797 – 799, 865 – 880 ’19 ].  If the nucleotides in the circle can reach across the circle and base pair with each other forming a duplex of any length, it will bind to PKR inhibiting it.  Most circular RNAs are expressed at only a handful of copies/cell, the cell containing just 10,000 of them.

The work found that overexpression of a single circular RNA able to form duplexes (dsRNA) inhibits PKR.  Over expression of linear RNA of the same sequence does not, nor does overexpression of circular RNA which can’t form dsRNA.

So when an invader with dsDNA or dsRNA gets into the cell, RNAase L, a cytoplasmic endonuclease is activated, cleaving circular RNA, and uninhibiting PKR.

So it’s back to the drawing board for mRNA and those parts (introns, 3’UTRs) we didn’t think were doing anything.  Perhaps that’s why there are so many of them, and why they take up more room in mRNA and genes than the ones coding for amino acids.  Also it’s time to look at RNAs as protein binders and modifiers, rather than the other way around as we have been doing.

Here’s a link to an earlier member of the series — https://luysii.wordpress.com/2019/04/15/forgotten-but-not-gone-take-ii/xa

Of what use is an inactive enzyme?

Why should a cell take the trouble make an enzyme protein with no enzymatic activity? It takes metabolic energy to store the information for a protein in DNA, transcribe the DNA into RNA and then translate the RNA into protein. Is this junk protein a la junk DNA? Not at all — and therein lies a tale.

All sorts of nasty bugs inveigle their way into cells, among them viruses (such as influenza) whose genome is made of RNA, rather than DNA. Not only that, but in many virus their genome is not single stranded (like mRNA) but double stranded with two RNA strands base paired to each other (just like DNA, except for an extra oxygen on the ribose sugars in the backbone).

Nucleated cells don’t contain much double stranded RNA (dsRNA) outside the nucleus, so it almost always means trouble. An extremely elegant mechanism exists to find and respond to such RNA. Recall that double helix molecules can reach enormous lengths.The 3.2 billion base pairs of our genome, if stretched out, would be more than a yard.

Well we have at least 4 genes which bind dsRNA and then signal trouble. They all make a molecule called 2′ – 5′ oligoadenyic acid (2-5A) from ATP, so they are called OligoAdenylate Syntheses (OASs). The 2-5A, once made wanders about the cell until it finds another enzyme called RNAase L. 2-5A binds to RNAase L causing it to dimerize and become active. RNAase L then destroys all the RNA in the cell, killing it along with the invading virus. Pretty harsh, but it’s one way to stop the virus from spreading and killing more cells.

A recent paper http://www.pnas.org/content/112/13/3949.full concerns OAS3, which has 3 catalytic modules rather than just one like most enzymes. Even worse, 2 of the 3 catalytic modules can’t make 2-5A (but they still can bind dsRNA). OAS3 is a large protein (over 1,000 amino acids), so it has some length to it. The 3 catalytic modules are spread out along OAS3 with the active catalytic module at one end and one of the inactive modules at the other.

The modules at both ends bind dsRNA, but only the active module makes 2-5A when it does. Interestingly, the inactive module binds dsRNA much more strongly than the active one.

OK, you’ve got the picture — what possible use is this rather Byzantine set up?

See if you can figure it out.

It’s incredibly clever and elegant, and shows the danger to regarding anything within the cell as functionless (or junk). Teleology rides supreme in molecular and cellular biology.

Give up?

OAS3 essentially acts as a molecular ruler making 2-5A only when long dsRNA (e.g. over 50 nucleotides long) binds to it. The inactive module gloms onto longish dsRNA, holding it tightly until till Brownian motion brings it to the other end of OAS3 activating the catalytic module to make 2-5A. This is good as the cell normally contains all sorts of shorter RNA duplexes (the binding of microRNAs to the 3′ end of mRNAs come to mind — but they are much shorter (22 nucleotides at most).