Billions of years ago, as simple lifeforms evolved toward complexity, a selfish genetic entity emerged, acting as a genome invader. Utilizing a copy-and-paste mechanism, this intrusive piece of code replicated relentlessly, embedding itself across various genomes.
Over time, this genetic trespasser, known as LINE-1, permeated all eukaryotic organisms, including humans, contributing to about one-third of our genome. Initially dismissed as junk DNA, recent revelations have unveiled its significance.
LINE-1’s aggressive integration into the genome can induce chaos, leading to disease-causing mutations. The orchestrator of this genetic invasion is a crucial protein called ORF2p. Understanding the intricacies of ORF2p’s structure and function opens avenues for exploring potential therapeutic targets across various diseases.
In a collaborative effort involving numerous academic and industry partners, scientists at Rockefeller University have achieved a breakthrough by capturing the high-resolution core structure of ORF2p for the first time. This achievement has illuminated novel insights into LINE-1’s mechanisms underlying disease, as detailed in the publication in Nature.
Senior author John LaCava, a research associate professor at The Rockefeller University, highlights the significance of this work in advancing rational drug design against LINE-1. The newfound knowledge holds promise for developing innovative therapies and strategies to combat diverse conditions, including cancer, autoimmune diseases, neurodegeneration, and other age-related ailments.
Evolutionary mates
LINE-1, a retrotransposon, is a mobile genetic entity capable of converting RNA back into DNA during replication, integrating itself into various locations within an organism’s genome. Within the realm of retrotransposons, LINE-1 is part of a broader category with diverse members, including endogenous retroviruses (ERVs) resembling HIV and Hepatitis B (HBV).
The evolutionary roots of LINE-1 are enigmatic, yet it shares an evolutionary connection with group II introns, ancient mobile elements dating back approximately 2.5 billion years. Over the course of 1 to 2 billion years, retrotransposons like LINE-1 have coevolved with their host organisms.
Co-first author Trevor van Eeuwen, a postdoctoral fellow in Rockefeller’s Laboratory of Cellular and Structural Biology, describes the ongoing battle between LINE-1’s attempts to insert itself and the host’s protective measures for its genome.
Our cells harbor millions of genetic fragments derived from LINE-1, with the majority being inactive remnants—a testament to unsuccessful attempts at hijacking the replication machinery. However, around 100 LINE-1s remain functional, and their impact is often detrimental. A recent study by LaCava, Michael P. Rout, and collaborators highlighted that cancer cells produce a protein called ORF1p, generated by LINE-1.
Although LINE-1 and its proteins have been subjects of study for over a decade, understanding has been hindered by the elusive nature of one of its proteins, ORF2p, which expresses infrequently. “LINE-1 has been challenging to study due to its peculiar features, such as an unusual replication cycle and the mysterious ORF2p protein that has eluded capture,” notes LaCava.
Collaboratively, Martin Taylor of Massachusetts General Hospital and Harvard Medical School, along with LaCava, overcame these challenges. Taylor’s pivotal work involved purifying the full-length ORF2p and a shorter “core” version crucial for L1 replication, paving the way for significant breakthroughs in understanding the intricacies of LINE-1 and its associated proteins.
Jack-of-all-trades
Employing a synergy of X-ray crystallography and cryo-EM, the research team unveiled two previously unknown folded domains within the core of ORF2p, shedding light on LINE-1’s capacity for self-replication.
ORF2p showcases structural adaptations finely tuned for diverse tasks, according to van Eeuwen. Serving as a versatile protein, it manages everything from replication to insertion, a capability that sets it apart. Remarkably, while most viruses rely on potentially hundreds of reverse transcriptase proteins for replication, ORF2p singularly executes these multifaceted functions.
However, when LINE-1 becomes active in the cytoplasm, it adopts a viral mimicry strategy. As van Eeuwen elucidates, “it generates RNA:DNA hybrids that mimic a viral infection when detected.” This mimicry provides a potential resolution to the enigma of how ORF2p triggers the innate immune system, contributing to conditions like autoimmune diseases.
The research discerned that interactions with genetic material in the cytoplasm trigger the cGAS/STING antiviral pathway. Consequently, this pathway prompts cells to produce interferons, activating the immune system and inducing inflammation—an analogous response to a viral infection.
van Eeuwen underscores LINE-1’s primary role in proliferating copies of itself, acknowledging the inherent risk of these sequences potentially disrupting genes. Yet, he also highlights the intriguing prospect that these movements could give rise to new genetic elements or novel functionalities that prove beneficial to the host.
The path ahead
Moving forward, the researchers aim to unravel the functions of the two recently discovered core domains, delving deeper into their intricacies. While pursuing this, van Eeuwen emphasizes that “our structural elucidation of ORF2p establishes a foundation for forthcoming investigations essential to unravel the LINE-1 insertion mechanism, its evolutionary trajectory, and its implications in disease.”
The team is also eager to explore the clinical implications of their discoveries. Given the connection between retrotransposons and retroviruses, the researchers tested HIV and HBV treatments in the study to assess their efficacy against LINE-1. However, these treatments proved ineffective, indicating the necessity for tailored therapeutics designed to address LINE-1’s unique attributes.
“This breakthrough paves the way for the rational design of improved LINE-1 inhibitors, and we anticipate these advancements will progress to clinical trials in the near future,” notes LaCava.
Rout underscores the broader significance of the study, stating, “This research highlights the potential of integrating diverse datasets and the expertise of multiple laboratories to unravel fundamental biomedical questions.” The collaborative effort showcases the power of synergy in addressing complex biological phenomena and advancing our understanding of critical genetic processes.
Source: Rockefeller University