MicroRNA – the gray eminence of the genome

What exactly is microRNA?

MicroRNA is a class of RNA that is very small in size. Mature microRNA molecules, also called ‘miRs’, are very short – only 22 nucleotides long. MicroRNAs can bind to other RNA molecules which are a matrix for protein production (hence the name: messenger RNA, or ‘mRNA’ for short). The mutual binding of both types of RNA depends on the sequence of these molecules, which makes their interaction specific and limited only to specific miRNA-mRNA combinations. When miRNA binds to mRNA, the production of the protein encoded by the mRNA is inhibited.
The Nobel Prize winners made their discovery in 1993. Earlier, in the 1980s, the mechanisms of transcription control, i.e. the process of reading genetic information – transcribing it from DNA to mRNA, were described. Further proteins, called ‘transcription factors’, were identified, which activated or inhibited transcription by binding to gene regulatory sequences.
At a time when most research on the regulation of gene expression was focused on finding more mechanisms of transcriptional control, Victor Ambros and Gary Ruvkunow described a new and surprising way in which cells can regulate protein production. The surprise was, among other things, where the miRNA sequences are encoded. Only a small part of our DNA contains information about the structure of proteins – vast areas of our DNA do not contain the ‘recipe’ for proteins. Little was known at that time about the function of these non-coding areas and few researchers were interested in them, but they constitute as much as 90% of our DNA. The identification of genes encoding miRNA – which are located in these ‘unnecessary’ regions of DNA – has shown us how much there is still to discover.

How did the Nobel Prize winners discover microRNA? By chance?

Victor Ambros and Gary Ruvkun studied gene functions. They conducted research on Caenorhabditis elegans, a common soil-dwelling nematode, about a millimeter long, that feeds on bacteria. Despite its small size, C. elegans has many specialized cell types (such as nerve and muscle cells) that are also found in more complex organisms. This nematode turned out to be a very good model for research, among other things, because it is transparent – therefore the development of each of its organs can be seen very clearly. Moreover, it develops very quickly – it takes less than three days for it to develop from an embryo to a mature egg-laying individual. In such an organism it is easy to study the functions of genes, the disabling of which gives an effect visible after just a few days and not after weeks or months, as is the case when working with other experimental models.
So, Ambros and Ruvkun, examining mutations in C. elegans, in the genes responsible for controlling the development of the worm’s organs, identified two genes that interacted with each other in such a way that one inhibited the other. However, it was not known how this happens. The scientists began to investigate this phenomenon more closely and discovered that one of the genes (lin4) encodes not a protein but RNA (today we would say – microRNA) that recognizes the mRNA encoded by the second gene (lin14), attaches to it and inhibits its action. Initially, it was suspected that this mechanism is unique to nematodes, but it soon turned out that it is also present in human cells.

What role does this microRNA play in the cell?

Gene expression is a multi-step process: DNA, in the transcription process, produces mRNA based on which protein is produced in the translation process. By binding to mRNA, miRNA disrupts the translation process, which affects the amount of protein produced. Therefore, miRNA controls the level of gene expression at a later stage than the already mentioned transcription factors. It is estimated that 60% of our protein-producing genes are under the control of microRNA molecules.

The Nobel Prize winners made their discovery 30 years ago and their work is said to have opened a new era in molecular biology. Is this really the case?

Their discovery truly sparked a boom in the search for microRNAs. Since then, scientists have demonstrated the existence of thousands of microRNA links to biological processes. However, as for the significance of the discovery made by Ambrose and Ruvkun, it would be an exaggeration to call it epoch-making. But it is extremely important for understanding how complex the processes that regulate gene expression patterns are, i.e. which of the 30,000 genes that each cell contains are active and produce proteins, and which are not. It is worth remembering that after synthesis of proteins is completed, they are further modified, which affects their functions. This means that the process of regulating the amount and activity of produced proteins is very complex and multi-level.
The complicated mechanism of regulating protein production at the transcriptional level is compounded by the influence of microRNAs. It should also be added that each mRNA may be subject to the influence of several microRNAs, and each microRNA affects several or several dozen different mRNAs. This means that the produced microRNA does not affect one specific target gene, but affects groups of genes containing the appropriate sequence. Regulation of gene expression with the participation of microRNAs is a concerto for many strings.

At what stage of understanding microRNA and its mechanism of action are we currently at?

The mechanism of action is already well known. The MicroRNA molecule is modified in many stages until – in a complex with a protein – it attaches to the appropriate mRNA, influencing its further expression. However, what followed the discovery made by the Nobel Prize winners was a massive wave of searches for new microRNA molecules. Since we know the human DNA sequence, it was possible to select microRNA coding areas using appropriate mathematical algorithms. Based on their nucleotide sequence, it is also possible to predict which genes they will affect. The studies that are currently being published concern mainly validation of these predictions and describe the role of this phenomenon in the course of many diseases. The results are sometimes very clear and show a connection between the disease and the presence of specific microRNAs in tissues or body fluids.

Does this mean that microRNA is already used in diagnostics?

Indeed, diagnostic tests using microRNA testing are already available. The list of diseases the diagnosis of which can be supported by this method includes, among others, thyroid cancer and pancreatic cancer. For example, such a test can determine whether a tumor is potentially malignant or not.
We also have diagnostic kits that allow us to find out what tissue the disease originates from, in cases where it is particularly difficult to determine.
However, it should be emphasized that the diagnosis of diseases is always based on results of many tests, the efficacy of which is well documented. MicroRNA tests are currently only a supportive tool.

Can knowledge about microRNA translate into new treatment methods?

MicroRNAs are molecules that are products of our genes. However, therapy uses drugs that resemble microRNA – they either have the same effect as normal microRNA or the opposite effect (they inhibit microRNA). Despite great hopes, treatment with these molecules turned out to be very difficult and, to date, none of these drugs has been registered, although several are in clinical trials. Unfortunately, there were cases where studies were interrupted due to serious side effects.
Therapy using microRNA is very difficult because these drugs are not very selective. The limitation is the route of administration and natural barriers that protect our cells from absorbing microRNAs circulating in the blood. A drug administered intravenously reaches all organs, although we would like it to target only the focus of the disease and act only there. Unfortunately, this does not happen.
This means that the drug inhibits not only diseased cells, but also healthy ones. The course of one of the clinical trials shows how huge a problem this is. It concerned therapy directed against solid tumors using synthetic microRNA 34a. It turned out during the study that the same microRNA 34a that helps destroy tumors is dangerous to cells of the lymphatic system. The drug did destroy cancer cells, but it also reached the spleen and bone marrow, severely damaging these organs.

If not yet in the treatment of cancer, for which diseases could microRNA have therapeutic applications?

The results of studies in the treatment of hepatitis C with a microRNA 122 antagonist are very promising. In this case, the drug administered into the vessels reaches the liver, i.e. exactly the organ affected by the disease, inhibiting the replication of the virus. This therapy is already in the second phase of clinical trials and has a good chance of being registered.
To sum up, the current state of affairs is that microRNA therapies carry a high risk of complications. There is great enthusiasm about them, but there is no confirmation in clinical trials yet, which this does not mean that this state of affairs cannot change in the future.

Interviewed by: Iwona Kołakowska
Fot.: Michał Teperek
Communication and Promotion Office