DICER: More than just an enzyme.

It slices, it dices, it’s found in quinces. Well actually, it has been found ubiquitously in eukaryotes – from plants to animals, fungi, and protists. Dicer is an enzyme that cleaves double-stranded RNA, which may sounds boring to you, but it is my favorite enzyme in the world. Why? It defends against viruses, particpates in a gene regulation system, and it is the basis for a process called RNAi, a promising tool for genetic research and crop biotechnology. But that’s not quite enough to qualify as a truly awesome macromolecule in my book, it also lends itself to a really nifty metaphor. Furthermore, recently completed research conducted at UC Berkeley has expanded our knowledge about how it works.

First, a little background.

Genes are instructions for making proteins, and are encoded in the double-stranded DNA in the nucleus of a eukaryotic cell. In order to make a protein from a gene, the code must first be transcribed onto a single strand of RNA called a messenger RNA, or mRNA. This leaves the nucleus and proceeds to a structure called the ribosome, which translates the instructions into a sequence of amino acids that make up a protein.

Viruses hijack these systems when making copies of themselves. Some insert their DNA into the chromosomes of the host cell, some create DNA strands from RNA that they insert into the cell, and finally, some insert mRNA molecules into the cell that go straight to the ribosomes to produce proteins. What is a cell to do to defend itself? Stop the mRNA before it can get to the ribosome.

Post-transcriptional gene silencing (PTGS) is a process by which cells stop mRNA molecules from making proteins, thus silencing them after transcription, but before translation. The process by which cells do this is called RNA Interference, or RNAi. It all starts with double-stranded RNA.

dsRNA

Although DNA is double-stranded, RNA is usually single-stranded in the cell. The 3-letter triplets, or codons, of the single-stranded mRNA correspond to the amino acids that end up in the final protein. Thus, this strand is called a sense strand. An antisense strand would be a sequence of letters that are the opposite sequence, which is attracted to the sense strand. This attraction is what keeps the two strand of DNA together, a mutual attraction based upon the complimentary sequences on either strand.

Sense mRNA: AUG GUG ACC UGA

Antisense RNA: UAG CAC UGG ACU

If you’ll notice, the A’s are opposite the U’s, and the C’s are opposite the G’s. Thus, just like in DNA, sense and antisense RNA strands attract each other and become double-stranded or dsRNA. But as soon as the cell finds dsRNA, along comes Dicer to chop it up, thus preventing it from ever forming a protein. What benefit does the organism get out of this?
Double-stranded RNA is sometimes formed by transposons, which are stretches of DNA that can self-replicate and move around in the genome, which can sometimes be disruptive. (Their movements cause the varied colors in indian corn, for example.) Secondly, dsRNA is also sometimes the genetic material of choice for viruses, and finally dsRNA is also used by cells to silence some of their own genes during development. We don’t know at this point whether Dicer evolved for combating viruses first, or for developmental reasons, but Dicer and Dicer-like (DCL) proteins are specialized for many dsRNA-chopping applications.

How does RNAi work?

The general mechanism of RNAi is pretty simple. When a strand of RNA is recognized as a target, it is chopped up and a piece of it is used to find other copies of it to destroy. You could think of RNAi as a sort of immune system based off of RNA processing, which wouldn’t be too far from reality, or even, the metaphor I will eventually reveal.

When Dicer encounters a dsRNA, it chops it up into little pieces of about around 20 base-pairs long, which separate into their sense and antisense strands. These are called “short (or small) interfering” RNA or siRNA. There’s that term “interfering” again. It won’t be the last!

Dicer then passes the baton to a protein complex called the RNA Induced Silencing Complex or RISC, which picks up the bite sized pieces of siRNA. Now the RISC + siRNA together can interfere with the process of translation. The siRNA provides a template that matches up to a copy of the original strand (a viral gene being replicated, for example), and when a match has been found, it is targeted for destruction. This system is very sensitive – it only takes a few molecules of dsRNA and the cell can effectively prevent matching RNA molecules from producing proteins. A protein called RdRp will also amplify– make copies of – the siRNA so that the cell doesn’t run out.

In addition, siRNAs have also been found going from cell to cell in plants and some animals, silencing genes at a distance. In the case of viruses, siRNAs can give other cells the heads-up about a virus before they encounter it themselves. In a very real sense, RNAi is a process by which cells can be vaccinated against intruders, or even their own genes..?

Yes, Dicer also dips its fingers into preventing a cell’s own endogenous genes from creating proteins. The mRNAs of some genes will fold back on themselves, a structure called a hairpin (for its similarity to bobby pins), forming a short stretch of dsRNA. Dicer may also chop these up, releasing more short strands of RNA, but these are instead called micro RNAs, or miRNA. A miRNA is pretty much the same thing as a siRNA, and loaded onto the protein complex RISC, is used to interfere with developmental genes. Micro RNAs make nifty ‘off-switches’ for developmental genes, and we are finding Micro RNAs involved in multiple developmental processes, from limbs to the brain – definitely an area of research to follow this year.

And finally, through its skillful chopping activity, Dicer helps to silence transposons. Also known as “jumping genes,” tranposons are stretches of DNA that reproduce within genomes, spreading from chromosome to chromosome, which can cause deleterious (harmful) mutations. So Dicer will pass off siRNAs to a group of proteins called the RNAi induced transcriptional gene silencing complex, or RITS, which will use the siRNAs to silence – turn off – the genes at the source.

A visual representation of the process

Now I have filled your head with all sorts of acronyms. You don’t need to remember them! In summary, Dicer chops double-stranded RNA into short pieces, which can come from viruses, transposons or jumping genes, and the cell’s own genes. These short pieces of RNA are used to recognize sequences in messenger RNA molecules or in DNA on chromosomes to interfere with them and therefore turn off unwanted genetic instructions.

Now, the new research.

The lab of Jennifer Doudna, UC Berkeley, recently completed their research on the molecular structure of Dicer, published on the Jan 13 issue of Science. You can download the paper from their lab website, here.

In short, they have pinned down the molecular structure of a Dicer protein that exists in Giardia intestinalis. They figured out the functions and arrangement of its parts, such as RNAse parts IIIa and IIIb, (Yellow and Green) which do the chopping, and the PAZ domain (orange) which binds to the RNA molecule. The dsRNA is shown in blue, and the purple dots are Eridium metal ions, which look to be the chopping sites. (Note how each pair straddles one of each of the strands.) But one interesting thing that they found was a connecting helix, shown in red, which acts as a ruler and determines how long the chopped RNA will be.

Isn't it Beautiful?
This is significant because the different sizes of chopped RNAs are often used for different functions. This alludes to more detail than I would like to put in here, but suffice to say this gives us a little peek into how Dicer proteins have evolved for all of their varied tasks, not to mention, shows us how we can engineer Dicers to cut dsRNA into any length we choose.

Okay, Mr. Scientist, but what can we do with all of this stuff?

Actually, we’ve already been putting this knowledge to good use. A large part of genetics involves breaking things – eliminating genes to understand their functions in cells. But this becomes problematic when you are dealing with genes that are essential for development, or living for that matter. But now with RNAi, geneticists can allow an organism to develop normally, and then inoculate its cells with dsRNAs that match specific genes. By the iron chef chopping of Dicer, the matching genes are turned off and the researchers can see the consequences – essentially opening a window into genetics that we’ve never had before.

But what is more remarkable is what genetic engineers have already been doing with RNAi. It is in many ways a kind of immune system, and so you can genetically vaccinate a cell against one of its own genes or those of a virus by inserting a copy of that gene (usually in reverse). The two RNAs meet up, form a dsRNA, and Dicer takes it from there. This basic idea is what saved the Hawaiian papayas.

The Papaya Ringspot Virus, or PRV, devastated the papayas in Hawaii. Breeding and selection could do nothing to stop it, and so University of Hawaii and Cornell University (NY) scientists decided to try to genetically vaccinate the papayas against PRV. All they needed to do was to engineer a piece of a PRV gene into the papaya, and Dicer took care of it. I could go on about how well it worked, but a picture is worth a thousand RNAs.Seeing is believing.

The square that you see in this picture is the patch of transgenic papayas that have been ‘vaccinated’ against PRV. The scraggly and stumpy disturbances around the square are the normal papayas.

Researchers in Japan have also made a genetically-decaffeinated coffee bean by the same process. Most decaffeinated coffee, if you dare to drink it, has a funny taste because of the methods they use to remove the caffeine. Well, now you can have all the taste, and only 25% of the caffeine, thanks to Dicer.

There are many things on the horizon that Dicer and its RNAi mechanism may be able to accomplish for us. Here’s a short list.

· dsRNA-based vaccines and/or treatments for viral infections, perhaps including HIV.Transgenic Petunia demonstrates suppression of endogenous pigment gene.

· Gene silencing of genes in cancer cells.

· Treatment of genetic diseases?

· More fantastic instances of viral resistance engineered into crops.

· Interesting floral pigment patterns, because after all, the first hints of the effects of RNAi were this:

Dicer as a Metaphor.

I am quite enamored with the genes-as-ideas metaphor. I will expound upon this elsewhere, but in brief, I consider science to be a reliable way of sifting through ideas, and pseudoscience, because it appears to be science but does not entail its rigors, to be a parasite of this process. Much like a virus is a parasite of a cell.

Oftentimes, you will come across articles about, and essays and statements from all sorts of pseudoscientific people and organizations. And sometimes intelligent people will help perpetuate them without recognizing it. What can we learn from Dicer?

Dicer stops unwanted genes from ever becoming proteins, and so in the genes = ideas metaphor, it follows that you can interfere with the replication of pseudoscientific and just-plain-wrong ideas. Just chop them up and pass out little tidbits to everyone else to help them recognize them when they see them! Just as Dicer chops up dsRNA molecules and passes the fragments to neighboring cells, so too shall I chop up the claims of pseudoscientists which you can use as mental vaccines against these ideas when they come around again. (Hey, I’m defining myself, ok?)It's Dicing Time!

This fits in with my overall theme of inoculating people against pathogenic thoughts. Whenever I decide to do a good chopping of bogus claims, look for the little picture of Dicer.

Dicer is more than just an enzyme – chopping up pathogenic instructions is a way of life!

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Karl Haro von Mogel

Karl Haro von Mogel serves as BFI’s Director of Science and Media and as Co-Executive Editor of the Biofortified Blog. He has a PhD in Plant Breeding and Plant Genetics from UW-Madison with a minor in Life Sciences Communication. He is currently a Postdoctoral Scholar researching citrus genetics at UC Riverside.