Molecular biology work carried out by the Molecular Neurobiology lab led by Damián Refojo at IBioBA, together with Manuel de la Mata’s team at the Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE), has revealed key information about the control that circular RNAs exert on gene expression. Studying these novel intracellular structures may be useful to explore the development of therapies against tumour, infectious and central nervous system pathologies.
By conducting studies in cell cultures, IBioBA researchers, in collaboration with IFIBYNE, analyzed a key role of molecules discovered a few years ago, called “circular RNAs”, in the regulation of gene expression. The study, published in the prestigious journal Nucleic Acids Research, contributes to understanding how these intracellular structures differ from the more “traditional” linear RNAs, which could be used in the future in the development of vaccines or to correct genetic alterations that occur in various diseases of the central nervous system, infectious or tumor diseases.
“Our work is part of a line of basic research. However, as is often the case in such risky, government-funded projects, basic research studies can lead, even unexpectedly, to valuable therapeutic technologies. The COVID-19 pandemic has taught us many things, not least that RNA technology – which has enabled the development of many vaccines (which have yielded billions of dollars for the companies that developed them) – will be a fundamental biological tool in
human and animal health for years to come”, says Manuel de la Mata, one of the study’s directors, a CONICET researcher at the Institute of Physiology, Molecular Biology and Neurosciences (IFIBYNE, UBA-CONICET) and professor at the Faculty of Exact and Natural Sciences at the University of Buenos Aires.
Damián Refojo, director of IBioBA (CONICET-Max Planck), who also led the breakthrough, says that “those who manage these technologies will be the ones who can best adapt to new therapies and even develop new therapeutic strategies, with the consequent benefits for human or animal health, but also for production and trade”.
Circular RNAs
In the 1960s, the coding ribonucleic acid (RNA) known as messenger RNA (mRNA) was discovered and acts as an intermediary between DNA and its end product, proteins, which are crucial for cell structure and function.
Genetic information “flows” from DNA to protein manufacture: DNA is first “transcribed” into mRNA form, which is then “translated” into protein form. There is a great diversity of coding RNAs that are translated into the various proteins in cells. However, since 2000, it has been discovered that there is a vast and complex world of RNA molecules that are not translated into proteins, but which also have essential functions for the cell. These are the so-called non-coding RNAs, which can be short linear molecules, long linear molecules or rarer species such as circular RNAs, which are the least understood at the moment.
What is it about circular RNAs that gives them their unique properties and what functions do they perform? These and other questions de la Mata and Refojo set out to begin to answer, and so they decided to conduct a joint study.
In 2015, the Refojo lab, by then at the Max Planck Institute of Psychiatry in Munich, published a collaborative study in the journal Molecular Cell, led by Dr Nikolaus Rajewsky of the Max Delbruck Center in Berlin, Germany, describing for the first time that these circular RNAs are highly expressed in the brains of different animals and even in humans.
“Although the function of most of these circular RNAs is unknown, they represent a fertile field of study. We now know that circular RNAs are more stable compared to linear RNAs and that some, in particular, have important functions in different cell types and tissues of the body. However, the mechanisms by which these circular RNAs exert their function are far from being understood”, says Refojo.
Moreover, the researchers wanted to collect data on the interaction between circular RNAs and other type of non-coding RNAs called microRNAs. “MicroRNAs are small molecules that are crucial role in regulating gene expression. They are known as ‘silencers’ of gene expression. They function by attaching to cognate sequences at the ends of messenger RNAs (mRNAs), inhibiting their translation into proteins or promoting their degradation”, explains de la Mata. He continues: “The function of ‘switching off’ specific genes is necessary for the normal functioning and development of organisms. Otherwise, different types of alterations take place and many of them can lead to disease”.
Likewise, the normal functioning of cells and the organism requires that, at a certain point, microRNAs must be eliminated once they have fulfilled their function through a process called TDMD (Target-directed microRNA degradation).
In this regard, de la Mata made a substantial contribution to describing the TDMD phenomenon during his postdoc at the Friedrich Miescher Institute (FMI) in Basel, Switzerland, leading to a scientific article in the journal EMBO Reports (2015) and other related work. “TDMD is a very relevant cellular process because by controlling the amounts of microRNAs it also controls the abundance of mRNAs and ultimately of the proteins present in the cell”, explains the researcher.
Circular RNAs and gene expression
The interaction between circular RNAs and microRNAs “is a particularly intriguing area of research: some circular RNAs can hijack microRNAs, neutralizing their ability to regulate other messenger RNAs and thereby affecting the gene regulatory network. This mechanism may have significant implications for various biological processes and diseases”, says de la Mata.
To reveal information about how circular RNAs and microRNAs interact, study’s authors conducted experiments on neurons obtained from the brains of mouse embryos and human cell lines. Using genetic engineering methods and bioinformatics analysis, the scientists found that multiple circular RNAs influence the stability of different microRNAs. “Our results support the notion that circular RNAs influence TDMD, i.e. the degradation process of specific microRNAs: in some cases they enhance the degradation process and in others they inhibit it. But a central point of our work was to show that two RNAs that have the same composition (i.e. the same nucleotide sequence or ‘letters’) and differ only in that one is linear and one is circular, can have different effects on TDMD, meaning that circularity itself can change the function of these RNAs”, says de la Mata.
“In light of these results, we will begin to explore potential therapeutic uses of these circular RNAs in various infectious, tumour and central nervous system diseases”, adds Refojo.
“Understanding these types of molecules (circular RNAs and microRNAs) has, in my opinion, two possible ramifications with enormous potential. The first is their use as tools in the biotech and pharmaceutical industries; interest in finding applications for both types of RNAs has exploded in recent years, ranging from circular RNA vaccine projects to microRNA-based treatments for certain types of cancer. The second has to do with improving the way we understand the regulation of gene expression in cells, whose potential utilities are infinite”, says Federico Fuchs, one of the first authors of the study who participated in the work while he was a CONICET doctoral fellow in the group led by de la Mata. Today, Fuchs works in the Department of Microbiology at Harvard Medical School in Boston, USA. He adds: “We can only ‘fix’ those things we understand, so knowing in greater detail new types of regulation can allow us to find the cause of diseases that have eluded us until now, or to choose new strategies to develop treatments”.
“Understanding the regulation of gene expression is fundamental to understanding biological systems. In this scenario, circular RNAs are becoming more and more talked about, especially because of their abundance and their role in the brain. By studying them in more depth, we can understand more details about the functioning of neurons, the brain and find clues to various diseases”, says Jerónimo Lukin, also first author of the paper while a doctoral fellow in the Refojo lab and now a scientist at the Icahn School of Medicine at Mount Sinai, New York, USA.
De la Mata and Refojo finally agree on one concept: “The development of circular RNA-based vaccines and the possibility of detecting these molecules in blood and urine as an early diagnostic system for neurodegenerative diseases and cancer has already begun to be explored, so understanding the properties that circularity confers on these RNAs will allow us to better manipulate these molecular tools in the future”.
The study involved collaborations with the laboratories of Jeremy Wilusz, from Baylor College of Medicine in the United States, and Gerhard Schratt from ETH-Zurich, Switzerland. With the latter laboratory, de la Mata and Refojo have obtained funding from the Swiss National Foundation (SNSF) to continue this line of research.