blog-from-the-dark-side

On a break

Thu, 13 Jan 2011 23:48:23 +0100

On a break over the summer (here in NZ)!
Will be back with more from the Dark Side in early Feb.

Happy New Year for 2011!!!!
Lesley

Catastrophe - RNA viruses on the brink

Sat, 06 Nov 2010 20:36:45 +0100

Fast RNA evolution – a recipe for disaster

RNA evolution is a fascinating topic. Molecular evolutionists have used RNA virus examples for many years because RNA evolves fast and displays many of the principles we are trying to get across to our students. Take for example the concept of ‘error catastrophe’. Error catastrophe is a wonderful phrase to pop into any science conversation. Basically it means that when the mutation rate is high, just a little more can push an RNA organism over the edge into extinction.

Alright, this sounds very Frankenstein mutant monster-ish but put in context, RNA viruses are organisms that use RNA as their genome and include HIV, Hepatitis, SARS, Rubella, Poliovirus and Measles. To succeed in replicating in their host (us) they need to overcome the host defences (like all that RNAi) so they mutate so the virus particles that have the best fitness will pass onto the next host. So within an infected host you do not have clonal (identical) copies of a virus but instead you have a population with minor differences between them. However, the down side to having high error rates that can permit high mutation is that if you go over the limit you produce too many errors and your genes fail. Mutation is good, so long as you still function.

Going over this limit (or error threshold) was investigated by Manfried Eigen in 1971 and there are some beautiful mathematical scratchings involved in calculating this limit based on mutation rate and genome size. Breathe a sigh of relief because I am not going to go into them. In short, unless you control your errors you can only sustain a genome size of about 200 nucleotides. To go beyond that genome size you need error checking enzymes and sacrifice the ability to mutate super-fast. Humans have 2.9 billion base pairs in their DNA and a lot of checking.

This brings up the idea of ‘if I mutate the hell out a virus, will it die’. The answer is...yes...for some.

A famous experiment by Crotty et al. used Ribavirin (an mutagen) on the Hepatitis C virus and showed that the action of this drug was to increase error and it did kill the virus (see the graph below). There is a lot of research around that looks at mutagens and viruses but you do have to watch out for what is happening to the host. If you take movies as your source of science you could say it was no good killing the virus if your host becomes something from ‘Splice’. RNA viruses really do live life on the edge, and despite all other life being against them, they persist. Not a happy thought as the northern hemisphere moves into cold and flu season. Sorry but as you sniffle you can always check up on Error Catastrophe and imagine mutating your invaders to extinction.

Crotty et al. 2001 PNAS 107:6895-6900

The graph shows that poliovirus populations exist near the edge of error catastrophe, as there is a rapid decline in RNA genome infectivity at levels of mutagenesis only slightly higher than normal. The LI50 (50% loss of specific infectivity) is the mutation frequency at which 50% of the viral genomes are lethally mutated, indicated by the dashed line. Wild-type poliovirus genomes contain an average 1.5 mutations/genome. 100mM ribavirin treatment caused about 1.9 mutations/genome, 400mM ribavirin caused about 6.9 mutations/genome, and 1,000mM ribavirin caused about mutations/genome.

SnoRNAs – tricky little suckers!

Sat, 30 Oct 2010 20:44:44 +0100

SnoRNAs – tricky little suckers

SnoRNAs (small nucleolar RNAs) are a class of small RNAs found in eukaryotes (like us) and archaea (those strange bacteria-like greeblies that live in extreme environments). They are about 100-200 nucleotides in length, come in a couple of flavours depending on their secondary structure and small motifs and can do a variety of things. In fact there are some snoRNAs that we just don’t know what they do. In general however, they modify or guide the modification other RNAs especially rRNAs, snRNAs and tRNAs. The modifications include pseudouridylation (try saying that after a few beers) and 2’-O- methylation. Basically the chemical structure of the nucleotide is altered slightly giving that nucleotide different chemical function. Nucleotide modification can give increased thermal stability to an RNA structure which is important since RNAs can be greatly affected by temperature. We still don’t know a lot about how these modifications and how they aid the cell, only that they do.

One group of snoRNAs are the C/D box snoRNAs, named after C and D motifs contained in the RNA. These snoRNAs bind to 4 proteins then bind to the target RNA to guide 2’-O-methyationAnother group of snoRNAs have H/ACA motifs and function to direct the conversion of uridines (U’s – the RNA equivalent to T in DNA) to pseudouridine. Just to make life more fun there are some snoRNAs that have both C/D and H/ACA motifs and function in both types of modification.

It is those snoRNA that do not have a target modification site that are gaining interest with researchers. Some C/D box snoRNAs are involved in the process of imprinting where only one copy of the gene from each parent is turned on. The copy to be turned off is targeted by the snoRNA, and promoter regions that turn the gene on are methylated so they don’t work. And it appears that these snoRNAs can get lonely. A whole cluster of snoRNAs containing 29 copies SNORD116 (HBII-85) and 48 copies of SNORD115 (HBII-52) has been implicated in Prader-Willi syndrome (a genetic disorder).

Not all snoRNAs guide modification but direct cleavage instead, and it is likely that as we investigate further we will find yet again that RNA-protein complexes do more than we think. We will likely find that there are even more RNA of all sizes (short, medium and long) connecting together in the tangled web of RNA networks. We can be comforted that these RNAs know what they are doing, even if as yet we don’t know all of what they do.

They are truly multifunctional molecules!

For some general information: http://en.wikipedia.org/wiki/Small_nucleolar_RNA

SnoPatrol: how many snoRNA genes are there? Paul P Gardner, Alex Bateman and Anthony M Poole http://www.biomedcentral.com/content/pdf/jbiol211.pdf

http://biochem.ncsu.edu/faculty/maxwell/Research.htm

How to find an RNA –or more precisely how to tell your computer to find an RNA

Sat, 23 Oct 2010 19:43:07 +0100

How to find an RNA –or more precisely, how to tell your computer to find an RNA

Now that I told you that finding a non-coding RNA gene can be difficult…in fact you can find some classes rather easily. It all comes down to computer programming. Computers are great. If you tell them what you want in clear and precise ways they will go out and find it and leave you to have a nice cup of coffee. However, it is up to you to tell them the right directions and that is the tricky part of finding RNAs. When we know what to look for –secondary structure and sequence motifs – we can find them. Take for example a transfer RNA, the non-coding RNA that delivers amino acids to the ribosome to convert the message in mRNA into proteins. It has a very clear secondary structure (often called the clover-leaf structure because of its shape – see the diagram below). The fun part is converting the human-readable structure into a code that the computer can look for, we do this using what is called ‘bracket notation’ where the paired nucleotides are represented by brackets and the unpaired by dots. If you go around the tRNA structure from 5’ end (on the right top) to 3’ (left top) the sequence and structure looks like this (with the bases that can change being called N and D stands for any nucleotide that is not a C):

GGACGUGUNGCGCGUAGDCGGDAGCGCNCUCCCUUGGCNNGGGAGAGGNCUCCGGTNCGAUUCCGGACUCGUCCACCA

(((((((..((((..........)))).(((((.......))))).....(((((.......))))))))))))....

You can see there are four regions of pairings (or stacks) represented by the open and closed brackets. These combinations can get very complicated but usually the computer software can work it out. Once you have this structure you are on the way to searching a long piece of DNA to see if that structure is contained within.

For those of you who would like to play a little with RNA structure head to the VARNA software site http://varna.lri.fr/demo.html (you will need to have an up to date version of Java installed for this to run). Remember if you change an opening bracket on one side of a stack then you will need to change the corresponding closing bracket. Try dragging the stacks around and have some fun!!!

http://www.wiley.com/college/boyer/0470003790/structure/tRNA/trna_intro.htm

Why didnt we know about RNA before?

Sat, 16 Oct 2010 20:41:05 +0100

Why are RNA functions so new to us?

This is a question I have been asked by many students. If RNA is so critical, and non-coding RNAs so common, such as those involved in regulation (RNAi), viral defence (siRNAs, CRSPRs) and the general running of the cell (RNase P, tmRNA), why are we only just learning about them?

The answer is that only recently do we have the knowledge to recognise that it is RNA doing these functions and the technology to uncover and research them. Researchers studied protein coding genes for decades because once we knew roughly how they change over time. If the nucleotide sequence (its string of G’s, T’s in DNA (U’s in RNA), C’s and A’s) changes then the protein sequence will be wrong and the protein may not work.

With non-coding ncRNA the nucleotide sequence is not forced to keep its patterns as it does for proteins. Instead the string codes for how the RNA folds and binds. This means that large portions of the sequence string can be completely different between species so long as the base pairing is the same, and small protein binding sequences are there. In RNA there is extra flexibility in the binding since G can bind to U as well as C, and other strange pairings such as G:A and C:A occur frequently in some molecules. Secondary structure rules also have some flexibility. As you can see in the figure below (I have tried to make it big enough) the RNase P RNA can form a different structure in humans and one of our parasites Giardia. The sequence is quite different and the structure different but the function is the same. RNA has a lot of flexibility which means that it has the potential to evolve to do more and new functions.

But this flexibility also means that finding these genes can be extremely hard. When we look at a new species we can have no idea what the real structure will look like until we find the gene. We usually have to guess, and then see what we get from our guesses. It becomes not so much detective work as with other genomics but a real needle in the haystack exercise. More on that next week in ‘How to find an RNA- 101’.

Adapted from Kikovska et al. 2007 PNAS 104:2062-2067 The RNA sequence starts from the left top overhang, goes around the molecule and back to pair with the beginning again. Pairings are shown by dashes except for the very long range and tricky pairing in the P4 region.

Bacteria have RNA too!

Sat, 09 Oct 2010 20:27:57 +0100

Bacteria have RNA too!

Up to now most of what I have explained has been from eukayotes which are species whose DNA resides in a nucleus (like us, plants and fungi). Since I have been helping my colleague Patrick with his book chapter on prokaryotic small RNAs I thought that might be a good topic for this week since I am finding them so fascinating. Prokaryote is the collective name for bacteria and archaea (archaea are the fun species that grow at high temperatures, high salt and can have some very unusual chemistries and we are still very much in the learning stage as to how they operate in extreme environments).

Non coding RNA in bacteria are sometimes called small RNAs. Just like in humans we have RNAs involved in viral defence (called CRISPRs) that take bits of virus to use against them, and just like in humans the viruses try to work around that. There are also many small RNAs involved in responding to cell stresses ( examples are temperature too hot, temperature too cold, too much sugar).

One sRNA I am becoming fond of is called transfer-messenger RNA (tmRNA) which is part a transfer RNA (tRNA) and part a messenger RNA (mRNA). Normally the mRNA threads through the ribosome and tRNAs bring amino acids to the ribosome to build up the coded protein. However, sometimes the ribosomes get stuck, usually because something has gone wrong with the mRNA and the code is just unreadable. This can tie up valuable resources in the cell so in bacteria, tmRNA detects a stuck ribosome, uses the tRNA part to get into the heart of the machinery, then uses its mRNA part to code a rescue sequence to unstick the ribosome and release the faulty mRNA. The faulty mRNA is degraded and the ribosome can get on with the next mRNA. This whole processes is called trans-translation.

As you can see below the way the tmRNA folds is very complicated as you would expect for an RNA that is doing the job of two. We are still in the early days of discovering how RNA helps the bacterial cell as it was not too many years ago that this was thought impossible. I guess RNA really did take over the world.

Thanks to Wikipedia for the images: http://en.wikipedia.org/wiki/Transfer-messenger_RNA

Global warfare - RNA against the viruses (which also happen to be RNA)

Fri, 01 Oct 2010 19:59:42 +0100

Global warfare in the Cell

Unlike the rest of us who love RNA, the general cell (bacteria, humans alike) does not like RNA at all. Why you ask (or the two of you who read my blog). This is because RNA is typically a signature of viruses which are at open war with the cell. Some common RNA-based viruses are Influenza, HIV, SARs, Polio, Rabies and Measles just to mention a few. Now any virus needs a cell to reproduce, it just cannot reproduce lots of ‘baby’ viruses in any other way except taking over the cell as its own private nursery. The cell responds by attacking any stray RNA it can find, and by protecting its own RNAs by marking them with proteins or chemical modifications. RNAi (RNA interference) is also used to protect the cell from viruses. A system well studied in plants (Virus Induced Gene Silencing, VIGS) has the cell taking the RNA strands as soon as they are detected and using them as siRNAs (small RNA strands from the last blog), so the virus-produced siRNAs find other virus RNAs, pair up with them forming double stranded RNA allowing the Dicer protein to come in and chop up the virus. However, this is a war. The virus fights back by trying to disable the RNAi system in the cell. It is a good thing for us, and for plants that we have multiple lines of defence. In mammals, long double stranded RNAs can trigger an interferon response to boost our natural immune systems.

Bacteria have an RNA-like mechanism to tackle their viruses (also known as bacteriophages). This is called the CRISPR system, where parts of viruses are used as templates for an RNAi-type search and destroy mission. With these RNA-based defence mechanisms found throughout all life it is likely that RNA-viral defence mechanisms are very ancient (like billions of years old!) Viruses must therefore be just as ancient because nature does not evolve ahead of time, it does not look forward. A viral defence mechanism evolved because there were viruses and those single celled ancestors of ours had to have some defence or they didn’t have progeny. So RNA is used in defence against RNA viruses even today, showing us in plain sight perhaps a molecular fossil of the ancient RNA world.

Bacteriophage http://thet2phage.com/thesongs.html Influenza http://science.thomsonreuters.com/pharma/h1n1/

RNA turns genes off

Sat, 25 Sep 2010 21:38:21 +0100

RNA turns genes off!

Right, turning genes off is just one of the things that RNA is very good at doing. This process is called RNA-interference (RNAi) because the small RNA interferes with the gene’s mRNA moving from the transcription to translation processes (see blog 1 for a refresher on this). Today I will explain one part of RNAi which is how microRNAs (miRNAs) kind of work.

Small RNAs are transcribed that fold back upon themselves in what is called a hairpin loop structure so you have a double stranded RNA with two sides that are complementary. The pairing generally works as follows: A’s bind to U’s, The C’s bind to G’s, but with RNA the G’s can bind to C and U, and U can bind to A and G to give a bit more flexibility.

Double-stranded RNAs are targeted in the cell by the DICER protein and chopped into 20 nucleotide long fragments. One of the fragments (the miRNA) is picked up by the RISC system (RNA-induced silencing complex) which then goes looking for the mRNA with a sequence matching the small miRNA sequence. When this gene’s mRNA is found the miRNA binds to the mRNA and another protein called ARGONAUTE in the RISC chops the mRNA. As a consequence the mRNA does not move to translation and the gene is ‘silenced’. Essentially the RISC uses the miRNA as the signal to find its target gene.This is only one way in that miRNAs silence genes and there are many variations on this theme. RNAi systems in general are found throughout eukaryotes (cells with a nucleus) and a similar system has been found in prokaryotes (something for a later blog). An exception is the yeast Saccharomyces cerevisiae (baker’s yeast) which has lost many of the RNAi proteins. How S. cerevisiae turns its genes off without using the standard RNA-based system is the subject of much research.

Unraveling the Mysteries of Small RNAs, Dianne S. Schwarz, Science Online feature http://www.sciencemag.org/feature/data/prizes/ge/2006/schwarz.dtl

RNAi is used in the cell for other gene regulation processes including epigenetics and virus protection. I will expand on each of these in further blogs.

Matzke MA, Matzke AJM (2004) Planting the Seeds of a New Paradigm. PLoS Biol 2(5): e133.

Early Examples of Gene Silencing in Transgenic Plants – what happens when you silence genes in petunias

So where is this RNA world and do I need to be a gamer to find it?

Fri, 17 Sep 2010 20:35:07 +0100

A Wii or computer game on the RNA world would be fantastic and I wish I was a game developer! How much fun would it be to control RNA molecules to fight invading viruses, but not destroy the cell in doing so…ok maybe this game would have too limited a distribution.

The RNA world is a term coined originally to describe a stage in the earliest development of life. Because RNA as a molecule can store genetic information (like DNA) and perform catalytic reactions (like proteins do), it is believed that this molecule kept life going before the establishment of the DNA-> protein system we see in today’s cells. Another fact supporting this idea is that DNA requires proteins to replicate, and proteins require DNA as a storage molecule; DNA and proteins need each other so neither alone could have been an effective genetic material. RNA can do it (experiments in the laboratory have shown this). The phrase 'RNA world' can be blamed on Walter Gilbert in 1986 but the concept of an early RNA stage of life has been around since 1968 (according to Wikipedia), but more realistically probably since molecular evolutionists put their heads together to look at early life from a non-protein angle.

Many textbooks forget that having RNA alone is a hypothetical concept. In reality, there would have been amino acids (the building blocks of proteins) and short peptides (a few amino acids but not catalytic) as well as metal ions and other chemical compounds. We can see the possible effect these compounds would have had on the evolution of the present genetic system. Non-coding RNAs today commonly use metal ions for stability and RNA is never alone in the cell; it always binds to proteins.

The term RNA world is also being applied to today’s cells since RNA plays a prominent role in cellular activities. Part of that may be a reflection on the ancient nature of many RNA-based mechanisms, or how quickly RNA can evolve (when compared to DNA and proteins). I will go into the reasons why RNA was for decades, the forgotten molecule and why it has come into prominence again in another blog.

Wonderful image of the RNA world by Julie Newdoll

The Beginners guide to RNA

Sat, 11 Sep 2010 01:00:39 +0100

Right, my original plan was to post some new and exciting research in RNA...until I got some comments that I needed to explain my research to those more 'scientifically challenged'.

OK I can do that.

Lets start with the basics like the central dogma of life. (See the diagram below) I don’t know who came up with the name but it sounds cool for the textbooks so it kind of stuck. DNA is our famous double stranded genetic storage material, all our genes are there, ready to go. It is our cellular library of information. When a gene (or book) is needed proteins make a single stranded copy of the DNA (photocopy perhaps) which is then sent to the Ribosome (a cellular machinery made of RNAs and proteins) to be used as the blueprints for proteins…or so they said last century. However, many more discoveries were made that showed that some genes didn’t get turned into proteins, they functioned as RNAs.

The way they can do this has to do with blobs. Proteins are essentially blobs of chemicals with a very defined 3D shape. This shape means that they can fit together in complexes, forming chemical bonds and breaking them. RNAs are also chemical blobs which can also fold into 3D shapes, can do the same sorts of things as proteins but not quite as efficiently. RNAs that can do catalytic reactions are sometimes called Ribozymes.

These early discoveries on non-coding RNAs (or ncRNAs) really opened a can of worms. In humans alone, there are many classes of RNAs of different sizes and shapes doing different functions. Short ones of about 21 nucleotides (strings consisting of nucleotide bases, Guanine (G), Adenine (A), Cytosine (C) and Thymine (T)), can interfere with other genes being translated into proteins, thus regulating them. Longer ones (100-200 nucleotides) bind with specific proteins and modify other RNAs chemically to control their function. Some very long ones have really important jobs in turning off that pesky spare X chromosome in females (we have two and do not need a double dose of ‘those’ enzymes).

There is a big RNA world out there hidden in the darkness of our cells. In other blogs I will go into some of these RNAs and how they work.

Further reading:

Good old Wikipedia has some good definitions and links: http://en.wikipedia.org/wiki/Non-coding_RNA