Periodic Reporting for period 1 - MitoPNP (Structural and functional studies of human PNPase in mitochondrial RNA metabolism)
Reporting period: 2021-09-01 to 2023-08-31
Every living organism requires energy to survive and reproduce. In humans, this vital energy is produced by tiny powerhouses called mitochondria, found in almost every cell. Apart from being energy providers, mitochondria participate also in many other crucial processes such as immune response, inflammation and regulation of cell death, therefore it is critical to understand the mechanisms controlling their functions. When these organelles encounter problems, it can lead to various diseases, including cancer, neurodegenerative conditions, and abnormal inflammatory responses.
Even though mitochondria play a crucial role in our cells, they have only a limited set of genetic instructions to operate with. To function at their best, many important proteins needed by mitochondria are actually made in different parts of the cell and then transported into these organelles. One such protein is polynucleotide phosphorylase (hPNPase), which is found mostly in the mitochondrial intermembrane space (IMS), located between two membranes that surround the central part of the mitochondria, known as the mitochondrial matrix. However, what exactly hPNPase does in the IMS is still a mystery. Some hPNPase can also be found in the mitochondrial matrix, where it partners with another protein,Suv3, and in a complex these two proteins cooperate in breaking down RNA. PNPase can also be found in bacteria, where it breaks down RNA, but when it teams up with another protein and specific RNAs, it changes its function: instead of degradative, it takes on a protective role. Interestingly, human and bacterial PNPases share a high degree of similarity in their genetic makeup and structure. This suggests that hPNPase might also have a dual mode of action.
My research aims at understanding the role of hPNPase, especially in the intermembrane space of the mitochondria. My working hypothesis is that hPNPase has two different ways of interacting with the RNA it encounters – one is to break it down, and the other is to protect it, and that the protective one might be especially important in the IMS. On the other hand, when hPNPase is in the mitochondrial matrix and in complex with its partner protein Suv3, it tends to be more focused on breaking RNA down. Therefore, by physical separation of hPNPase in two different compartments, both its activities could operate in the mitochondria to support organelle function.
To better understand human PNPase I started by cloning the gene encoding it into a special genetic construct. This allowed me to produce the protein in bacteria. Then, I isolated the protein from the bacterial culture and purified it using various chromatography techniques. This highly pure form of the enzyme was then ready for further study, including its biochemical and structural analysis. Next, I have prepared some of hPNPase substrates: RNAs that were reported to interact with the enzyme inside our cells. I delved into how hPNPase interacts with and degrades various RNAs to figure out what sequences or structures in RNA might control these interactions. This analysis helped me select the most interesting complexes for further structural studies.
To unveil the three-dimensional structure of hPNPase and its interactions with RNA, I used a cutting-edge technique called electron cryo-microscopy (cryoEM). This is a rapidly advancing method and is used by scientists all over the world to gain insights into complex macromolecular assemblies. This advanced technology is essential for modern protein research and is not yet fully established in Poland. It can be used for purposes like designing new drugs, investigating genetic mutations that cause diseases in patients, or gaining insights into the functions of medically significant proteins. I successfully prepared samples for cryoEM and collected data that resulted in high-resolution maps of hPNPase both on its own and when bound to various substrates. Now I'm in the process of creating 3D models based on these maps to better understand how this enzyme functions in relation to its structure. My research helps us to determine if hPNPase can function as an RNA regulatory factor as well as an RNA degrading enzyme in the mitochondria, what would have implications for better understanding of the inner workings of mitochondria, and this in turn could help develop effective treatments for mitochondrial disorders.
Even though mitochondria play a crucial role in our cells, they have only a limited set of genetic instructions to operate with. To function at their best, many important proteins needed by mitochondria are actually made in different parts of the cell and then transported into these organelles. One such protein is polynucleotide phosphorylase (hPNPase), which is found mostly in the mitochondrial intermembrane space (IMS), located between two membranes that surround the central part of the mitochondria, known as the mitochondrial matrix. However, what exactly hPNPase does in the IMS is still a mystery. Some hPNPase can also be found in the mitochondrial matrix, where it partners with another protein,Suv3, and in a complex these two proteins cooperate in breaking down RNA. PNPase can also be found in bacteria, where it breaks down RNA, but when it teams up with another protein and specific RNAs, it changes its function: instead of degradative, it takes on a protective role. Interestingly, human and bacterial PNPases share a high degree of similarity in their genetic makeup and structure. This suggests that hPNPase might also have a dual mode of action.
My research aims at understanding the role of hPNPase, especially in the intermembrane space of the mitochondria. My working hypothesis is that hPNPase has two different ways of interacting with the RNA it encounters – one is to break it down, and the other is to protect it, and that the protective one might be especially important in the IMS. On the other hand, when hPNPase is in the mitochondrial matrix and in complex with its partner protein Suv3, it tends to be more focused on breaking RNA down. Therefore, by physical separation of hPNPase in two different compartments, both its activities could operate in the mitochondria to support organelle function.
To better understand human PNPase I started by cloning the gene encoding it into a special genetic construct. This allowed me to produce the protein in bacteria. Then, I isolated the protein from the bacterial culture and purified it using various chromatography techniques. This highly pure form of the enzyme was then ready for further study, including its biochemical and structural analysis. Next, I have prepared some of hPNPase substrates: RNAs that were reported to interact with the enzyme inside our cells. I delved into how hPNPase interacts with and degrades various RNAs to figure out what sequences or structures in RNA might control these interactions. This analysis helped me select the most interesting complexes for further structural studies.
To unveil the three-dimensional structure of hPNPase and its interactions with RNA, I used a cutting-edge technique called electron cryo-microscopy (cryoEM). This is a rapidly advancing method and is used by scientists all over the world to gain insights into complex macromolecular assemblies. This advanced technology is essential for modern protein research and is not yet fully established in Poland. It can be used for purposes like designing new drugs, investigating genetic mutations that cause diseases in patients, or gaining insights into the functions of medically significant proteins. I successfully prepared samples for cryoEM and collected data that resulted in high-resolution maps of hPNPase both on its own and when bound to various substrates. Now I'm in the process of creating 3D models based on these maps to better understand how this enzyme functions in relation to its structure. My research helps us to determine if hPNPase can function as an RNA regulatory factor as well as an RNA degrading enzyme in the mitochondria, what would have implications for better understanding of the inner workings of mitochondria, and this in turn could help develop effective treatments for mitochondrial disorders.
I have cloned and optimised expression and purification conditions of human PNPase, successfully obtaining pure and homogenous protein suitable for further biochemical and structural studies. In order to investigate hPNPase activity and binding specificity, I have also prepared several RNA substrates. To this end, I have cloned the genes encoding specific human RNAs and used them as templates in in vitro transcription reactions. Produced RNA was purified and tested in degradation and binding assays with the enzyme. In order to understand the structure-function relationship of hPNPase I have used electron cryomicroscopy (cryoEM) to determine the structure of the apo enzyme, as well as its complexes with different RNAs, including one substrate that was interacting with hPNPase but was not getting degraded. I am finishing data processing and model building for all five datasets collected during the project.
Publications disseminating the results are expected to be published in the future, once all the works are completed. The results of the project were presented at several conferences and meetings, including ISMB symposium and EMBO YIP RNA Structural Sectorial Meeting. They were also featured during multiple outreach activities, like Science Festival or interviews for the radio and a magazine.
The project allowed me to improve my scientific skills by learning new techniques and strengthening the understanding of the ones I already know. I have established myself as a local cryoEM expert and helped others to successfully implement cryoEM in their research. I have also tremendously improved my soft skills regarding project management, presentation, organization and leadership. I have started formation of my own research team and recruited a PhD student and two undergraduates, as well as secured funding sufficient for the project completion. In conclusion, the project allowed me to achieve professional maturity and advance my career towards independent position.
Publications disseminating the results are expected to be published in the future, once all the works are completed. The results of the project were presented at several conferences and meetings, including ISMB symposium and EMBO YIP RNA Structural Sectorial Meeting. They were also featured during multiple outreach activities, like Science Festival or interviews for the radio and a magazine.
The project allowed me to improve my scientific skills by learning new techniques and strengthening the understanding of the ones I already know. I have established myself as a local cryoEM expert and helped others to successfully implement cryoEM in their research. I have also tremendously improved my soft skills regarding project management, presentation, organization and leadership. I have started formation of my own research team and recruited a PhD student and two undergraduates, as well as secured funding sufficient for the project completion. In conclusion, the project allowed me to achieve professional maturity and advance my career towards independent position.
According to our knowledge we have for the first time visualized full length human PNPase and hPNPase-RNA complexes, and we hope the complex of hPNPase with the RNA that is not degraded by the enzyme will be the first one to prove its dual function. Such discovery will significantly improve our knowledge about the mechanisms regulating human mitochondria, and could impact on future treatment of mitochondrial diseases.
Moreover, through a bilateral transfer of knowledge the project formed the basis for the formation of a new research team and introduction of the cryoEM at the host department. The expertise in this structural biology technique strengthened by this project will support medically-relevant and high-technology-based research at the host institution. The outreach activities the Fellow actively participated in contributed to promotion of the interest in science and scientific careers. All of the above should positively impact both on the excellence of science and its societal engagement, on a local and broader level within the European Research Area.
Moreover, through a bilateral transfer of knowledge the project formed the basis for the formation of a new research team and introduction of the cryoEM at the host department. The expertise in this structural biology technique strengthened by this project will support medically-relevant and high-technology-based research at the host institution. The outreach activities the Fellow actively participated in contributed to promotion of the interest in science and scientific careers. All of the above should positively impact both on the excellence of science and its societal engagement, on a local and broader level within the European Research Area.