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Description
My research plan aims at understanding the role and function of hPNPase in the intermembrane space of the mitochondria. My working hypothesis is that hPNPase displays two modes of action on its RNA substrates, and the protective one could be exploited functionally in the IMS, where the enzyme would be RNA-mediated regulator of mitochondria function. On the other hand, in the mitochondrial matrix hPNPase in complex with Suv3 would always act in a degradative mode (Figure 1). This way, by physical separation of hPNPase in two different compartments, both its activities could be supporting optimal performance of the mitochondria.
I would like to conduct biochemical and molecular biology experiments that will determine if hPNPase can function as an RNA regulatory factor as well as an RNA degrading enzyme. I also plan to determine the precise three-dimensional structure of hPNPase using cryo-electron microscopy, a rapidly evolving technique allowing scientists all over the world to gain insight into different complicated macromolecular assemblies. My planned research will help us understand the role of human PNPase in the mitochondrial intermembrane space, and with such knowledge we could be one step closer to recognise causes of some mitochondrial disorders.
Summary of project results
Mitochondria, small organelles present in nearly every cell, have long been regarded as the powerhouses of the cell due to their essential role in energy production. However, in recent years, scientists have discovered that mitochondria are far more than energy generators—they are critical signalling hubs involved in numerous vital cellular processes, including cell death. This makes understanding mitochondrial function an important scientific goal.
The mitochondrial genome encodes only a small number of proteins essential for mitochondrial function, with the majority of proteins being imported from the cytoplasm. Among these is polynucleotide phosphorylase (PNPase), an evolutionarily conserved enzyme best characterized in bacteria. In bacterial cells, PNPase is primarily known for its role in RNA metabolism, where it acts as an exoribonuclease, degrading superfluous RNA molecules. Additionally, PNPase can function as an RNA polymerase, adding nucleotides to RNA 3’
end. Recent studies also revealed that bacterial PNPase can serve as an RNA chaperone when interacting with Hfq, a bacterial RNA-binding protein, and small non-coding RNAs (sRNAs).
Given the significant similarity between bacterial and human PNPases, it is plausible that human PNPase also fulfils multiple roles within mitochondria. These could include not only RNA degradation but also other RNA-regulatory processes. The aim of my project was to explore the roles of human PNPase in mitochondrial RNA metabolism, focusing on identifying functions beyond its established role in RNA degradation in the mitochondrial matrix.
The project aimed to investigate the RNA and protein partners of hPNPase directly in human cells. My goal was to optimize a pull-down procedure to isolate hPNPase complexes from human mitochondria. These complexes would then undergo proteomic and RNA sequencing analyses, providing a comprehensive list of proteins and RNAs that co-purify with hPNPase from mitochondria.
To perform the pull-down experiment, I began by isolating mitochondria from human cell cultures. This was achieved through gentle cell lysis followed by differential centrifugation. Mitochondria are enclosed by two membranes, creating two distinct subcompartments: the intermembrane space (IMS) and the matrix. The purified mitochondria were fractionated into IMS fractions and mitoplasts, which contain proteins and nucleic acids from the mitochondrial matrix. Next, both whole mitochondria and the subcompartment fractions were subjected to a pull-down assay using a resin coated with anti-hPNPase antibodies.
These antibodies specifically bound hPNPase and its associated complexes present in the fractions. The presence of hPNPase in the final sample was confirmed by Western blot.
The initial experiments were conducted on a small scale to optimize each step of the procedure and confirm its overall success. Once optimized, the experiment was intended to be scaled up to obtain sufficient material for the identification of proteins and RNAs interacting with hPNPase. This approach would provide a detailed view of the hPNPase interactome in human mitochondria, advancing our understanding of the enzyme’s functions and its role in mitochondrial biology.
Due to unforeseen challenges in experimental procedures and multiple instances of failure of the equipment critical to the project execution, the original goals of the project could not be fully accomplished. To advance our understanding of hPNPase role in mitochondria, I concurrently investigated some protein and RNA partners of hPNPase that had been previously described in the literature. I prepared hPNPase complexes for structural studies and characterized its activity toward specific RNA substrates, as well as in the presence of its potential protein partners. Some of the results contributed to a collaborative manuscript focusing on another protein involved in mitochondrial RNA biology.
The implementation of the POLS project significantly enhanced expertise in cryo-EM techniques within Poland, equipping staff and students at the University with advanced skills to further utilize and develop these invaluable resources and knowledge. Although the determination of the precise function of hPNPase, an ancient and conserved enzyme, remains an open challenge, this project has made meaningful contributions by addressing some gaps in our knowledge of RNA metabolism in human mitochondria. These findings will be of interest not only to researchers and the scientific community but also to industry professionals and expert users who may find the results of this research applicable in their fields.