German Scientists identify Enzyme PUCH Which Can Help In Preventing Inclusion Of Parasitic DNA In Human Genomes Due To Pathogenic Infections

Genomics-Epigenetics: In the ever-evolving battle between our bodies and harmful pathogens, scientists have made a groundbreaking discovery that promises to shed light on how our cells combat internal threats posed by parasitic DNA. Researchers from the Institute of Molecular Biology (IMB) in Mainz, Germany, and the Max Perutz Labs in Vienna, Austria, have unveiled a newfound enzyme called PUCH. This enzyme plays a pivotal role in preventing the inclusion of parasitic DNA, known as transposable elements (TEs), into our genomes. The implications of this discovery extend far beyond its origins in the roundworm C. elegans, as it may provide a deeper understanding of the mechanisms our bodies employ to detect and fend off pathogenic infections.


The sequence of the synthetic piRNA precursor used in the assay. The red line indicates the expected cleavage position. Both the precursor and expected cleavage product were run in the two left-most lanes of every gel to mark where these molecules are expected. b, In vitro cleavage assay of the piRNA precursor using anti-GFP immunoprecipitated material from BmN4 cell extracts. Cells were transfected with eGFP–TOFU-2, TOFU-1, SLFL-3 or SLFL-4 at various combinations, as indicated. All observations were performed at least in duplicate. nt, nucleotides. c, Cleavage assays with recombinant minimal PUCH (mini-PUCH) and different RNA substrates. E216A indicates the presence of TOFU-2 containing the catalytic E216A mutation. All observations were performed at least in duplicate. d, RNA obtained from a cleavage reaction (using either wild-type or TOFU-2(E216A)-mutant mini-PUCH) was ligated to a 10-nucleotide-long 5′OH-containing RNA adapter. The ligation product is indicated by an arrow. The experiment was performed in triplicate. e, In vitro cleavage assay on different types of RNA substrate using the PUCH complex retrieved from BmN4 cells by immunoprecipitation (IP). All observations were performed at least in duplicate. f,g, Cleavage assays with mini-PUCH and the indicated substrates. The experiment was performed in triplicate for f and once for g.

The study findings are also extremely relevant in the current COVID-19 era where the SARS-CoV-2 virus and its spike proteins are affecting the DNA of the human host in a variety of ways.
 
The Genome's Hidden Battlefront
Our cells are under perpetual siege from an array of external threats, including viruses and bacteria. To safeguard against infections, our immune system deploys a complex network of cells and molecules designed to detect and eliminate these intruders. However, what is often overlooked is the constant battle occurring within our own genome. Approximately 45 percent of our genome consists of repetitive DNA sequences called transposable elements or TEs. These TEs have no defined function but are akin to genomic parasites that can copy and paste themselves into various locations within our DNA. This mobility poses a significant risk as it can lead to mutations, causing cells to malfunction or even become cancerous. In essence, nearly half of our genome engages in an ongoing guerrilla war, where TEs strive to proliferate while our cells strive to curb their spread.
 
The Ingenious Genomic Defense System
How do our cells combat these internal enemies? Fortunately, our cells have evolved a genomic defense system composed of specialized proteins tasked with hunting down TEs and thwarting their replication. Professor Dr René Ketting at IMB and Dr Sebastian Falk at the Max Perutz Labs report their groundbreaking discovery of the PUCH enzyme, a previously unknown component of this genomic defense system. PUCH's critical role lies in producing small molecules called piRNAs (PIWI-interacting RNAs), which act as sentinels, detecting TEs when they attempt to transpose. Once identified, piRNAs activate the genomic defense system, halting TEs before they can insert themselves into new locations within our DNA.
 
The PUCH Discovery
The Genomics-Epigenetics researchers uncovered PUCH while investigating the cells of the roundworm C. elegans, a widely-used model organism in biological research. However, the implications of this discovery extend well beyond this humble worm, offering insights into our own immune system. PUCH possesses a distinctive molecular structure known as Schlafen folds, which sets it apart. These folds are not unique to PUCH and are also found in mice and humans, where they seem to play a role in innate immunity, the first line of defense against viruses and bacteria. Some Schlafen proteins, for instance, interfere with viral replication in humans. Intriguingly, certain viruses, such as monkeypox viruses, may employ Schlafen proteins to subvert the cell's defense system. This suggests that Schlafen proteins might have a conserved role in immunity across various species, including humans.
 
Professor Ketting remarked, "Schlafen proteins may represent a previously unknown molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control TEs. This raises the possibility that Schlafen proteins serve as a common defense mechanism against both external threats like viruses and bacteria and internal ones, such as TEs. “
 
Dr Falk added, "This discovery may profoundly impact our understanding of innate immune biology."
 
The Mechanism of piRNA Processing
The discovery of PUCH marks a significant milestone in our understanding of piRNA biogenesis. PiRNAs are instrumental in controlling transposons, particularly in the germ cells, and their formation involves several key steps. One of these steps is the processing of piRNA precursors, a critical determinant of specificity in the genome immunity system. While the details of this processing have remained elusive, PUCH has emerged as the key player in initiating piRNA processing in C. elegans.
PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), is responsible for executing 5' end cleavage of piRNA precursors. Remarkably, PUCH-mediated processing is highly specific, requiring a 7-methyl-G cap (m7G-cap) and a uracil at a specific position. This specificity not only aids in substrate selection but also safeguards trans-spliced mRNAs from the PETISCO-PUCH machinery. PETISCO, a cytoplasmic protein complex consisting of PID-3, ERH-2, TOFU-6, and IFE-3, plays a crucial role in piRNA precursor stabilization and production. However, PETISCO itself lacks nucleases, leaving the question of which nuclease mediates 5' precursor processing and how it interacts with PETISCO open for future exploration.
 
Implications for Genome Immunity
The identification of PUCH as a ribonuclease comprising three subunits, each with an SLFN-like domain, signifies a significant expansion of the piRNA biogenesis toolkit in C. elegans. Although unrelated to the enzyme Zuc, which initiates piRNA biogenesis in mammals and flies, PUCH shares similarities in function, particularly in cleaving piRNA precursors at a specified distance from the 5' end. While Zuc depends on PIWI proteins binding to precursor 5' ends, PUCH relies on the presence of a 5'-m7G-cap, likely bound by PUCH itself. This unique specificity adds an additional layer of substrate selection, differentiating piRNA precursors from other cellular RNAs.
 
The discovery of PUCH also reveals intriguing connections between Schlafen-like domains and immunity. In mice, the Slfn gene cluster has been linked to immunity, exhibiting high rates of sequence divergence. Specific haplotypes of the Slfn gene cluster have been associated with parental incompatibility syndromes. Furthermore, SLFN proteins in humans have been shown to have various immune-related functions, from restraining translation of viral proteins during HIV infection to interfering with the replication of certain DNA viruses. These findings suggest a deep evolutionary conservation of SLFN-like domains in immunity and stress-related mechanisms.
 
Conclusion
The discovery of the PUCH enzyme represents a significant breakthrough in our understanding of how our cells defend against genomic parasites. Beyond the world of C. elegans, this discovery offers a fresh perspective on the broader mechanisms of innate immunity shared across species, including humans. PUCH, with its unique Schlafen folds and role in piRNA biogenesis, hints at a deeper connection between immunity and deeply conserved RNA-based processes that control transposable elements. As we continue to unravel the mysteries of the genome's hidden battlefront, the PUCH discovery may pave the way for new avenues of research and therapeutic interventions in the fight against infectious nucleic acids.
 
The study findings were published in the peer reviewed journal: Nature
https://www.nature.com/articles/s41586-023-06588-2
 
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