Thursday, 22 January 2009

Meaning out of nonsense

Imagine you are a eukaryotic cell. You have many genes and make many mRNAs. One problems is mutated genes, pseudogenes and mistakes by RNAPII all make mRNAs which contain Premature Stop Codons (PTC). These then make truncated proteins which could be toxic to the cell. Eukaryotes have evolved a mechanism to recognise these and degrade them. I guess prokaryotes do not have an analogous system (that I know of) because the half-life of these mRNAs are minutes long not hours long like most eukaryotic mRNAs. Eg the average mRNA half-life in Arabidopsis is between 4-6 hours and is very variable between mRNAs. Personally I am not convinced this is an important enough reason to have a quality control check mechanism. The system I am referring to is called Nonsense-Mediated mRNA Decay (NMD). Mediators of it have been found in the early branching Giardia and is conserved between fungi/animals to plants. A PTC is recognised as wrong rather than a correct stop codon at the end of a ORF during the first round of translation by the ribosome. When the ribosome reaches a stop codon it has two choices, terminate correctly or stall and cell effectors of NMD to take it away for degradation by whatever nucleases that cell uses. It was first found in yeast and the mutants were called upf1-3 (for up-frame shift 1-3) and these yeast grow fairly normally but have increased levels of transcripts with stop codons. Raises the question why did it evolve if not very important when you KO it. In C. elegance SMG1-7 were identified having genital defects. Strangely, these were involved NMD with SMG2-4 corresponding to UPF1-3. Homologues of most of these proteins are found in many other eukaryotes. UPF1 is generally phosphorylated when ribosome stalling takes place and condemns it to degradation. Interestingly, it is a DEAD box RNA helicase (SF2 I believe).

The two questions I find interesting is 1) how do organisms tell the difference between a PTC and normal stop codon and 2) how this can be used to regulated gene expression in cell signalling. You may ask ‘James, you charismatic stallion, how can something degrading useless mRNAs be useful or regulated for controlling gene expression’ and I would reply ‘wait and read the rest of this post you $#£!&%’. The first question is rather interesting as different organisms do different things. Yeast and invertebrates such as C. elegance and the fly pushers Drosphilia use something called the 3’ faux model. Something in the 3’ end allows normal termination when the stop codon is here but NMD starts when the stop codon (PTC) is distant from the 3’ end. It has been found the distance from the poly-A tail and therefore the Poly-A Binding Protein (PABP) determines this. When a long 3’ UTR is present the mRNA is degraded. When PABP is tethered close to the stop codon termination occurs normally. This also works in plants and mammals. However, another mechanism also works in both of them. When an introns is spliced out an Exon-Junction Complex (EJC) is left behind and if a EJC is found near a stop codon by the ribosome NMD starts. Why this operates in both plants and mammals but not yeast and invertebrates is curious. Did both mechanisms operate in the last common ancestor of plants and animals and has been lost a few times in the fungi and animal kingdoms or is it convergent evolution? I hope to better understand that.

Another and perhaps more important question is what is NMD role in plants and animals. Does it provide another function other than keeping toxic protein levels low? Short answers, yes. It affect development in C. elegance and embryo lethality in mice. Clearly it plays a role in animal development. In yeast the cells senesce sooner in upf1-3 mutants because telomeres shorten. In mammals, amino acid deletion causes problems for the ribosome and NMD is down regulated and genes for amino acids biosynthesis are up-regulated so more amino acids will be made. In Arabidopsis, when upf-1 is completely KO we see embryo lethality but Knock-down alleles see some developmental phenotypes (such as in the flowers) and altered stress responses.

I will finished up now but feel free to ask questions. I have just finished writing a 4-page grant proposal on this. Just to piss Mel off, I will say this. I didn’t do any reading for this. This was all from memory when I got home. No PDF files were opened – not one!

Thursday, 1 January 2009

Arch nemesis

Evidence is the cornerstone of science. It is what allows scientists to reject or accept hypotheses, and a robust piece of evidence can force the staunchest defender of a theory to completely reject it. People tend to like evidence when it supports their "beliefs", and dislike it when it refutes them, but scientists have no choice, they need to be apathetic and disinterested towards any results, so as to avoid introducing bias. However, scientists are also (rumoured to be) people, and the tendency to like or dislike a piece of evidence can be quite hard to resist.

How could a scientist maintain an interest in their research while avoiding interest in the data generated from it? It seems almost self-contradictory. What goals do scientists usually have when carrying out research? For a scientist working at a pharmaceutical company (assuming they wanted to keep their job), this goal would be to get a drug on the market. For a graduate student, the goal could be to find something surprising or revolutionary in order to jump-start their career. For a hardened senior professor, it could be to prove their own hypotheses right and boost their reputation (and maybe get in line for a Nobel prize?). All these goals are fundamentally selfish, and you would be hard pressed to find pure altruism in even the "most moral" scientist (whatever that means...). Perhaps that's because scientists tend to be ambitious, competitive animals, and that's a selfish motivator in itself, but I would say it's because people's goals tend to be selfish in nature.

These selfish goals scientists have certainly pose the most danger in making them introduce bias into their data. However, the drive that these goals give to scientists is what has led to so many historical discoveries. I'm sure some discoveries had altruistic motives but let's just say Watson and Crick weren't really thinking about saving humanity from terrible doom. Not to paint a sad picture of scientists, but sometimes the only thing that scientists get up for in the morning is the tiny chance that today's experiment will get them even slightly closer to achieving their goal.

I think that scientists manage to minimize the danger of introducing bias by finding the one person on Earth they most disagree with and establishing an intimate, life-long professional relationship with them, manifested mostly through heckling at conferences and angry e-mail exchanges. This works much more efficiently in academia than in industry, because industry scientists are usually either unwilling or not allowed to talk to outsiders about anything they do. Academics are also secretive to a certain extent in order to avoid being scooped, but the spirit of collaboration and criticism is much stronger. This kind of mutually abusive relationship is what every scientist needs, and I personally can't wait to meet my arch nemesis!

P.S. Happy 2009!