Saturday, February 4, 2012

The RNA World: knocking down FGFR3 in achondroplasia

This article is a sequel of the last one: I recommend you to read it first!

“There are more things between heaven and earth, Horatio, than thy vain philosophy can dream”, said a stressed Hamlet in the famous Shakespeare’s masterpiece. Never this so famous quote has been as current as now, at least for Science.
While researchers dig deeper and deeper to unveil the complexities of life, tons of new information are retrieved and our world turns richer – and more complicated. This is the very truth to someone who starts to look how intricate is the control of the genes.

The chemical world works like Lego

Before we continue, let´s establish a concept that may help the understanding of the complexities of the interaction among these so many biomolecules. With thousands of proteins and nucleic acids working together, anyone can become overwhelmed just trying to remember by heart the alphabet soup which describes the names of all these compounds.
So, let´s try to put this simple: look at these so many molecules making their reactions by coupling in a miriad of combinations, like we do with lego blocks (Figure 1). 

Figure 1. Tons of Lego blocks, tons of molecules.

Of course, although Lego blocks have many different shapes, you will have to use your imagination and think about much more shapes indeed to make justice to the biomolecules. Some of these blocks may have larger pins, others will combine only with triangles, light yellow round blocks will combine only with dark yellow ones, and so on.
When we speak about nucleic acids (DNA and RNA), the other term used to describe the way, for instance, DNA exists in the classic double strand chain (Figure 2), is complementarity. In this case, just to refresh, the four nucleotides that form DNA are combined like this: A links to T and C links to G. If one of the strips of the DNA chain (let’s call it sense strand) is constituted by ATG CGA, the other strip (the anti-sense strand) must be TAC GCT. We then say that TAC GCT is complementary to ATG CGA.  If we speak about RNA, the T is substituted by an U.

Figure 2. DNA structure
(from Annenberg Learner)
The result of mechanically combining lego blocks in specific modes will lead to beautiful figures or characters. Millions of combinations of proteins and/or nucleic acids determine chemical reactions through the transfer of electric charges to result in proteins or other molecules which will, in the end of the day, make what we are. The richness of life is given by the uncountable number of these combinations. This is just amazing.

RNA interference

In the last article, we started to learn about the many species of RNA which take part in the regulation of all aspects of the cell functioning. These RNAs are classified as non-coding RNAs, or ncRNAs, because they don’t give origin to proteins. Instead, they regulate or control one or more aspects of the cell nucleus machinery responsible for activating or deactivating the expression of the genes into proteins.

We saw that small molecules of RNA, made of about 21 nucleotides and called micro RNAs (miRNA), are capable of blocking the translation of proteins, the process by which the most known type of RNA, the messenger RNA (mRNA), is ‘read’. If the mRNA cannot reach the ribosome and be read, there will be no protein. To this mighty power, scientists gave the name of RNA interference.

The miRNAs are very important and theoretically, if we could identify one of them with a specific action only in the mRNA which encodes (carry the information needed to produce) the protein fibroblast growth factor receptor 3 (FGFR3), half the way would be done. The issue is that miRNAs are specific to their target mRNAs, but they regulate several targets at the same time, those which bear the sequence of nucleotides which complements the one in the miRNA which gives its powerful ability. As there is no currently known natural miRNA exclusive for FGFR3, the existing ones are still of no use as a therapy for achondroplasia (ACH).

The miRNAs are the tip of the iceberg for there are several other families of RNAs, We will look now at the other types of RNAs which could have a role in the control of the production of FGFR3.

The first group of these ncRNAs is called small interfering RNAs, or siRNAs. The siRNAs are as small as the miRNAs and they share quite the same mechanism of action. Both molecules are originally double stranded (they are combined in two complementary strips in the same way DNA is).

One of the mechanisms controlling the production of proteins is ruled by a complex of proteins called RISC. There is a place within this RISC complex where miRNAS and siRNAS can couple. When complete, the RISC binds to a mRNA. If the miRNA or the siRNA within the RISC complements to a specific part of the mRNA, the RISC stops there and attracts another enzyme that degrade the mRNA, thus hindering the production of the protein that mRNA encodes. Look at this animation of RNA interference provided by the journal NATURE. It will help you to understand the process.

The shape of miRNAs makes them difficult to be introduced from the exterior. Several enzymes called nucleases are present in the blood stream, in the cytoplasma and in the cell nucleus. They are a formidable barrier to deliver pieces of RNA to the cells because of their affinity with nucleic acids. Nucleases are extremely important for our defense, as they degrade pieces of nucleic acids which could be part of an invader organism (a virus, etc.).

To overcome this natural limitation researchers have already introduced a large number of chemical modifications in the RNA molecules to enhance their resistance to the nucleases, but miRNAs are less amenable than siRNAs to these modifications. Can we build a siRNA-like molecule? The answer is Yes. Some siRNAs have already entered clinical trials and a couple have been approved for use in a few indications.

Furthermore, FGFR3 has already been the target of experiments with siRNAs. Take a look in this recent study published by Drs. Laurence Legeai-Mallet and Jose Pintor, where they explore RNA interference in ACH chondrocytes. This work is a proof of concept that we can use siRNA to reduce FGFR3 expression. This opens the possibility of testing siRNA in an appropriate mouse model of ACH to see how the response is. The great challenge here is to make the siRNA reach chondrocytes as we discussed above. We will speak about this in another article.

The research in the ncRNA mechanisms also showed another characteristic of these molecules that can be used to repress the synthesis of proteins. A particular conformation some species of RNA adopt can block the reading frame of a mRNA. This conformation, which for the scientists who discovered them looked like a hair pin, is literally called hairpin. Short hairpin RNAs (shRNA) have been extensively used in the labs to let the researchers learn how a gene works. Using shRNAs (or, of course, other kinds of RNA interference, such as in Dr. Pintor’s work) they can learn the functions of a specific gene through the effects caused by the absence of the protein it expresses.

If you want to learn more about RNA interference, follow these links to broad and rich reviews on this compelling theme:
We have very briefly reviewed some characteristics of the mechanisms involved in RNA interference, an exclusive function some kinds of ncRNAs have. We still have a RNA species to visit (in the next article).

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