Billions of years ago, as primitive life forms became more complex, a selfish genetic component became a kind of colonizer of the genome. Using a copy-and-paste mechanism, this pernicious piece of code replicated and inserted itself repeatedly into a variety of genomes. Over time, all eukaryotic organisms inherited the code – including us. In fact, this ancient genetic element wrote about a third of the human genome – and was considered junk DNA until relatively recently.
This genetic component is known as LINE-1, and its aggressive intrusion into the genome can wreak havoc, leading to disease-causing mutations. A key protein called ORF2p enables its success – meaning that understanding the structure and mechanics of ORF2p could illuminate potential new therapeutic targets for a variety of diseases.
Now, in collaboration with more than a dozen academic and industrial groups, Rockefeller scientists have rendered the protein’s core structure in high resolution for the first time, revealing a host of new insights into LINE-1’s key disease-causing mechanisms. The results were published in Nature.
“The work will facilitate the rational design of drugs targeting LINE-1 and could lead to new therapies and strategies to combat cancer, autoimmune diseases, neurodegeneration and other diseases of aging,” says senior author John LaCava, research associate professor at Rockefeller University. .
LINE-1 is a retrotransposon, a kind of mobile genetic code that translates RNA back into DNA as it replicates and writes itself in different locations in an organism’s genome. There are different types of retrotransposons, including endogenous retroviruses (ERVs), which resemble HIV and hepatitis B (HBV).
The origin of LINE-1 is unclear, but it has an evolutionary link to group II introns, a class of ancient mobile elements dating back about 2.5 billion years. Retrotransposons like LINE-1 have been evolving with their host organisms for 1 to 2 billion years.
“It’s an ongoing battle between LINE-1 trying to insert itself and the host protecting its own genome,” says co-author Trevor van Eeuwen, a postdoctoral fellow in the Rockefeller Laboratory of Cellular and Structural Biology.
Millions of genetic fragments derived from LINE-1 are found in our cells. The vast majority are dormant evolutionary relics, evidence of failed attempts to hijack the replication machinery. But about 100 LINE-1s are operational – and generally not useful. A protein produced by LINE-1, known as ORF1p, is produced by cancer cells, as described in a recent study by LaCava, Michael P. Rout and their collaborators.
LaCava and Martin Taylor of Massachusetts General Hospital and Harvard Medical School have collaboratively studied LINE-1 and its proteins for more than a decade, but because ORF2p is expressed so low and infrequently, it has remained poorly understood. “LINE-1 has been very difficult to study because it has very strange characteristics,” says LaCava. “For example, it has an unusual replication cycle and the ORF2p protein that no one has been able to capture. But Marty and I finally reached a point where our research was mature enough that we could begin to study its structure.”
Taylor has made important advances in purifying the full-length ORF2p as well as a shorter “core” version that facilitates L1 replication; These advances facilitated the advances that followed.
Jack of all trades
Using a combination of X-ray crystallography and cryo-EM, the research team discovered two new folded domains within the core of ORF2p that contribute to LINE-1’s ability to make copies of itself.
ORF2p has structural adaptations uniquely suited to these endeavors, says van Eeuwen. It’s a sort of jack-of-all-trades protein, capable of handling everything from replication to insertion. But while most viruses need potentially hundreds of reverse transcriptase proteins to replicate, ORF2p does it all.
However, when LINE-1 is activated in the cytoplasm, “it acts like a viral mimic. It creates RNA:DNA hybrids that look like a viral infection when detected,” notes van Eeuwen. This viral mimicry suggests a possible solution to the enigma of how ORF2p activates the innate immune system, contributing to autoimmune diseases and other conditions. Her research found that interactions with genetic material in the cytoplasm activate the cGAS/STING antiviral pathway. In turn, this pathway causes cells to produce interferons, stimulating the immune system and leading to inflammation, analogous to what happens during a virus infection.
“Its main function appears to be to proliferate copies of itself, and as LINE-1 moves sequences around, there is a chance that those sequences will break a gene,” he says. “But there is also a chance that they will create new genetic elements or new functionalities that are beneficial to the host.”
The path ahead
In the future, researchers will seek to resolve the two newly discovered core domains and understand their functions. Meanwhile, “our structural elucidation of ORF2p lays the foundation for future studies needed to dissect and improve our understanding of the mechanism of LINE-1 insertion, its evolution and its roles in disease,” says van Eeuwen.
They also want to explore the potential clinical applications of their findings. As there is a relationship between retrotransposons and retroviruses, in the current study they tested treatments for the HIV and HBV retroviruses to see if they would inhibit LINE-1. They did not do so, suggesting that therapeutic design will have to be adapted to the unique characteristics of LINE-1.
“The work opens the door to rational drug design with better LINE-1 inhibitors, and we hope these will lead to clinical trials soon,” says LaCava.
And, as Rout adds, “this study also highlights the potential of integrating multiple types of data – and the expertise of multiple labs – to solve fundamental biomedical questions.”
This research project was an international collaboration. In addition to the Rockefeller scientists, crucial contributions were made by ROME Therapeutics, University Medical Center Groningen (J. LaCava laboratory), MSKCC (B. Greenbaum laboratory), Rutgers (E. Arnold laboratory), University of Alberta (M. Götte laboratory) , Dana Farber (K. Burns lab), UCSF (A. Sali lab), MPI Tübingen (O. Weichenreider lab), and UT Arlington (S. Christensen).