The death of the synonymous codon – V

The coding capacity of our genome continues to amaze. The redundancy of the genetic code has been put to yet another use. Depending on how much you know, skip the following four links and read on. Otherwise all the background you need to understand the following is in them.

https://luysii.wordpress.com/2011/05/03/the-death-of-the-synonymous-codon/

https://luysii.wordpress.com/2011/05/09/the-death-of-the-synonymous-codon-ii/

https://luysii.wordpress.com/2014/01/05/the-death-of-the-synonymous-codon-iii/

https://luysii.wordpress.com/2014/04/03/the-death-of-the-synonymous-codon-iv/

There really is no way around the redundancy producing synonymous codons. If you want to code for 20 different amino acids with only four choices at each position, two positions (4^2) won’t do. You need three positions, which gives you 64 possibilities (61 after the three stop codons are taken into account) and the redundancy that comes along with it. The previous links show how the redundant codons for some amino acids aren’t redundant at all but used to code for the speed of translation, or for exonic splicing enhancers and inhibitors. Different codons for the same amino acid can produce wildly different effects leaving the amino acid sequence of a given protein alone.

The latest example — https://www.pnas.org/content/117/40/24936 Proc. Natl. Acad. Sci. vol. 117 pp. 24936 – 24046 ‘2 — is even more impressive, as it implies that our genome may be coding for way more proteins than we thought.

The work concerns Mitochondrial DNA Polymerase Gamma (POLG), which is a hotspot for mutations (with over 200 known) 4 of which cause fairly rare neurologic diseases.

Normally translation of mRNA into protein begins with something called an initator codon (AUG) which codes for methionine. However in the case of POLG, a CUG triplet (not AUG) located in the 5′ leader of POLG messenger RNA (mRNA) initiates translation almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF) called  POLGARF (POLG Alternative Reading Frame — surely they could have come up something more euphonious).

Not only that but the reading frame is shifted down one (-1) meaning that the protein looks nothing like POLG, with a completely different amino acid composition. “We failed to find any significant similarity between POLGARF and other known or predicted proteins or any similarity with known structural motifs. It seems likely that POLGARF is an intrinsically disordered protein (IDP) with a remarkably high isoelectric point (pI =12.05 for a human protein).” They have no idea what POLGARF does.

Yet mammals make the protein. It gets more and more interesting because the CUG triplet is part of something called a MIR (Mammalian-wide Interspersed Repeat) which (based on comparative genomics with a lot of different animals), entered the POLG gene 135 million years ago.

Using the teleological reasoning typical of biology, POLGARF must be doing something useful, or it would have been mutated away, long ago.

The authors note that other mutations (even from one synonymous codon to another — hence the title of this post) could cause other diseases due to changes in POLGARF amino acid coding. So while different synonymous codons might code for the same amino acid in POLG, they probably code for something wildly different in POLGARF.

So the same segment of the genome is coding for two different proteins.

Is this a freak of nature? Hardly. We have over an estimated 368,000 mammalian interspersed repeats in our genome — https://en.wikipedia.org/wiki/Mammalian-wide_interspersed_repeat.

Could they be turning on transcription for other proteins that we hadn’t dreamed of. Algorithms looking for protein coding genes probably all look for AUG codons and then look for open reading frames following them.

As usual Shakespeare got there first “There are more things in heaven and earth, Horatio, Than are dreamt of in your philosophy.”

Certainly the paper of the year for intellectual interest and speculation.

The Bach Fugue of the Genome

There are more things in heaven and earth, Horatio,
Than are dreamt of in your philosophy.
– Hamlet (1.5.167-8), Hamlet to Horatio

Just when you thought we’d figured out what genomes could do, the virusoid of rice yellow mottle virus performs a feat of dense coding I’d have thought impossible. The following work requires a fairly sophisticated understanding of molecular biology which the articles in “Molecular Biology Survival Guide for Chemists” might provide the background. Give it a shot. This is fascinating stuff. If the following seems incomprehensible, start with –https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and then follow the links forward.

Virusoids are single stranded circular RNAs which are dependent on a virus for replication. They are distinct from viroids because viroids need nothing else to replicate. Neither the virusoid or the viroid were thought to code for protein (until now). They are usually found inside the protein shells of plant viruses.

[ Proc. Natl. Acad. Sci. vol. 111 pp. 14542 – 14547 ’14 ] Viroids and virusoids (viroid like satellite RNAs) are small (220 – 450 nucleotide) covalently closed circular RNAs. They are the smallest known replicating circular RNA pathogens. They replicate via a rolling circle mechanism to produce larger concatemers which are then processed into monomeric forms by a self-splicing hammerhead ribozyme, or by cellular enzymes.

The rice yellow mottle virus (RYMV) contains a virusoid which is a covalently closed circular RNA of a mere 220 nucleotides. A 16 kiloDalton basic protein is made from it. How can this be? Figure the average molecular mass of an amino acid at 100 Daltons, and 3 codons per amino acid. This means that 220 can code for 73 amino acids at most (e.g. for a 7 – 8 kiloDalton protein).

So far the RYMV virusoid is the only RNA of viroids and virusoids which actually codes for a protein. The virusoid sequence contains an internal ribosome entry site (IRES) of the following form UGAUGA. Intiation starts at the AUG, and since 220 isn’t an integral multiple of 3 (the size of amino acid codons), it continues replicating in another reading frame until it gets to one of the UGAs (termination codons) in UGAUGA or UGAUGA. Termination codons can be ignored (leaky codons) to obtain larger read through proteins. So this virusoid is a circular RNA with no NONcoding sequences which codes for a protein in either 2 or 3 of the 3 possible reading frames. Notice that UGAUGA contains UGA in both of the alternate reading frames ! So it is likely that the same nucleotide is being read 2 or 3 ways. Amazing ! ! !

It isn’t clear what function the virusoid protein performs for the virus when the virus has infected a cell. Perhaps there aren’t any, and the only function of the protein is to help the virusoid continue existence inside the virus.

Talk about information density. The RYMV virusoid is the Bach Fugue of the genome. Bach sometimes inverts the fugue theme, and sometimes plays it backwards (a musical palindrome if you will).

It is unfortunate that more people don’t understand the details of molecular biology so they can appreciate mechanisms of this elegance. Whether you think understanding it is an esthetic experience, is up to you. I do. To me, this resembles the esthetic experience that mathematics offers.

A while back I wrote a post, wondering if the USA was acquiring brains from the MidEast upheavals, the way we did from Europe because of WWII. Here’s the link https://luysii.wordpress.com/2014/09/28/maryam-mirzakhani/.

Clearly Canada has done just that. Here are the authors of the PNAS paper above and their affiliations. Way to go Canada !

Mounir Georges AbouHaidar
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Srividhya Venkataraman
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Ashkan Golshani
bBiology Department, Carleton University, Ottawa, ON, Canada K1S 5B6
Bolin Liu
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Tauqeer Ahmad
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and