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For the last 5 years, Richard Stefl and his lab mates have been studying the mysteries of non-coding RNAs (often called genomic dark matter), which so far, represents the missing link in understanding cell mechanisms and human diseases. Their research was supported by the prestigious ERC Consolidator Grant provided by the European Research Council. This type of grant is awarded to outstanding projects that have a chance to push human knowledge forward in a fundamental way. Richard Stefl, who leads a research group at CEITEC, managed to reveal structural insights into the control mechanisms of pervasive transcription and the biogenesis of non-coding RNAs.
Genomic dark matter is a high-sounding term for non-coding RNAs (ribonucleic acids). Non-coding RNAs are abundant and functionally important RNA molecules that are not translated into proteins. It seems that non-coding RNAs are responsible for many regulatory mechanisms inside of cells, and form a basis for the functioning of the human body as a whole. A defect, mis-regulation, or malfunction of non-coding RNAs is thought to contribute to serious diseases, including cancer. Thus, non-coding RNA expression profiles are valuable biomarkers in various human pathologies, and the targeting of non-coding RNA species has become a promising therapeutic approach for the treatment of human diseases. Stefl´s team was trying to map how this still mysterious genomic dark matter was created and its functions.
The analogy to dark matter known from the universe came from the enigmatic nature of genomic dark matter and its power to control mechanisms inside of the cell. Genomic dark matter was discovered only recently, with the help of new technologies, and due to a fresh and deeper knowledge about non-coding RNA. Originally, it was believed that RNA is only a primitive molecule that merely carries genetic information, but today, we know that the most important enzyme in the body is the RNA enzyme, which synthesizes proteins. It seems that RNA has a regulatory function as well, as it works from above and controls everything from the background. In his ERC project, Richard Stefl suggested an original approach to the structural mapping of dark matter formation. Researchers believe that a better understanding of the entire process would provide crucial information for molecular biologists, and could help them to better target the treatment of various serious illnesses.
Richard Stefl´s lab is affiliated with CEITEC Masaryk University. The team uses nuclear magnetic resonance (NMR) spectroscopy, electron microscopy, and other biophysical and molecular biology techniques to study interactions of biomolecular machines made of nucleic acids and proteins. The team’s main aim is to gain structural insights into the assembly and function of RNA processing and degradation machineries. The laboratory is mainly concerned with the chemical communication of molecules, but from a biological perspective. The researchers are keen to understand the structural basis behind this fascinating biological phenomenon.
Richard Stefl´s research group benefits from several types of state-of-the-art technologies that are available under one roof at CEITEC (MU), and from the expertise within CEITEC’s very strong structural biology program. The Stefl Lab collaborates with universities such as Oxford University and the Université de Paris. The ERC grant enabled the research group to expand its research focus from simple biomolecular complexes and small individual domains, to larger biological machineries like nucleosome and RNA polymerase as a whole.
We interviewed Richard Stefl about his newest discoveries concerning the structure of genomic dark matter, challenges he encountered, lessons he learned, and his research vision and future plans.
What did you discover during the past five years?
Using integrative approaches of structural biology, we have determined a number of structures that are involved in the transcription termination and metabolism of non-coding RNAs. These structural data provide a strong foundation for the deep analysis of these fundamental biological mechanisms. For example, we have revealed the structural basis for how these regulatory factors assemble and exchange on the C-terminal domain (CTD) of RNA polymerase II (Pol II) in a phosphorylation-dependent manner, and how they interact with RNA. We have also discovered new regulatory factors that associate with Pol II CTD, and have showed how these factors promote liquid-liquid phase separation of RNA polymerase II, an important phenomenon that dynamically controls the partitioning of transcription machinery in the nucleus.
How has your discovery challenged or enriched the current state of the given field?
RNA polymerase II timely recruits hundreds of regulatory factors in the course of the transcription process, which is orchestrated by phosphorylation marks in its C-terminal domain. RNA polymerase II is, in a way, decorated by these phosphorylation marks that create so-called CTD code. Our structural data revealed how this CTD code is read by the regulatory factors. We also showed that phosphorylation coupled to the cis-trans isomerization in the CTD functions as a molecular switch that regulates the association of some regulatory factors with RNA Polymerase II. We envisage that such a mechanism in which isomerases (important enzyme for cellular processes) control the timing of isomer specific protein-protein interactions is an important element of the CTD code. Most importantly, we found that regulatory factors, such as the NNS complex involved in the termination and processing of non-coding RNA, contains sequences that can mimic the CTD motifs. This opens up the exciting possibility that the dynamics of factors interacting with the CTD throughout the transcription cycle are regulated not only by the enzymes responsible for CTD modifications, but also by competitive interactions with proteins containing CTD-like motifs that mimic the particular CTD-modification pattern.
What was the greatest challenge/obstacle that you faced during project implementation?
Determination of the structure of weak protein-protein complexes is challenging and involves many biochemical and biophysical issues. The greatest challenge was that many of the studied complexes displayed a large degree of flexibility, which is challenging for the main structural biology technologies, such as X-ray crystallography.
How did you manage to overcome those obstacles?
To account for the flexibility of the studied systems, we employed integrative structural biology, which combines data from multiple experimental techniques to generate complete structural models. This approach offers great potential to linking a detailed atomic structure with cellular context, and consequently with disease. We took advantage of the high-end technologies of integrated structural biology available in the CEITEC Core Facilities, and fully utilised them to address the challenges of this project.
What are your professional plans for the future? In which direction will your research go?
All of our thinking begins with structural and mechanistic principles, with the ultimate goal of real clinical impact. We will continue to investigate Pol II CTD interactome and its ability to undergo phase-separation to form membrane-less organelles. In the nucleus, these condensates are thought to organise the stages of the transcriptional cycle and to facilitate critical biomolecular interactions.
ABOUT RICHARD STEFL
Associate Professor Richard Stefl was born in 1975. He graduated from Masaryk University in 1999, was a visiting student at the University of California in Los Angeles from 2000 to 2001, and defended his PhD dissertation in Physical Chemistry in 2001. From 2002 to 2006, he worked as a postdoctoral student at ETH in Zurich. Since 2007, he has been a Research Group Leader at CEITEC Masaryk University. He received an Associate Professorship in Biomolecular Chemistry at his alma mater in 2013. In 2015, he received the ERC consolidator grant entitled, “DECOR: Dynamic Assembly and exchange of RNA polymerase II CTD factors,” and was awarded by the Rector of Masaryk University for exceptional results in an international grant competition. In 2015, he received the President’s Prize from the Czech Science Foundation for best project and the Neuron Prize in chemistry. This prize is awarded to first-class scientists for their impact on the state of knowledge in biology.