Cryo-ET Reveals Retrotransposon Replication Inside Cells

An ongoing arms race is taking place within human cells: Transposons—also known as jumping genes or mobile genetic elements—can replicate and reintegrate into the genome, posing a threat to genomic stability by causing DNA rearrangements and mutations. To counter this, host cells have developed sophisticated defense mechanisms to prevent transposons from mobilizing.

For the first time, researchers have captured a retrotransposon in action within a living cell. By refining Cryo-Electron Tomography (cryo-ET) techniques, scientists imaged the retrotransposon copia in the egg chambers of the fruit fly Drosophila melanogaster at sub-nanometer resolution.

The international research team behind this breakthrough includes three scientists affiliated with the Vienna BioCenter: Sven Klumpe, currently at Jürgen Plitzko's laboratory at the Max Planck Institute of Biochemistry in Martinsried, who will soon join IMBA and IMP as a Joint Fellow; Julius Brennecke, a Senior Group Leader at IMBA, the Institute of Molecular Biotechnology of the Austrian Academy of Sciences; and Kirsten Senti, a staff scientist in the Brennecke group. Their collaboration also involved Martin Beck's group at the Max Planck Institute of Biophysics in Frankfurt. The findings were published in Cell.

Cryo-ET is a powerful imaging technique that enables researchers to visualize cellular structures in three dimensions at molecular resolution. The method involves capturing a series of 2D images from different angles and reconstructing them into a detailed 3D model. While cryo-ET has provided unprecedented insights into cellular ultrastructure, it has primarily been applied to unicellular organisms due to the need for rapid vitrification—freezing that prevents ice crystal formation. Multicellular tissues, which require high-pressure freezing, are often too thick for standard cryo-ET techniques.

A Capsid Resembling Retroviral Structures

In this study, the researchers used cryo-lift-out, an advanced technique that integrates focused ion beams and micromanipulation at cryogenic temperatures to prepare complex tissues for cryo-ET imaging. By working with Drosophila egg chambers and isolating individual cells, they resolved the structure of the copia retrotransposon’s capsid at a resolution of 7.7 Å—the first instance of a retrotransposon being visualized at sub-nanometer resolution in its native cellular environment.

Leveraging AI-driven structure prediction tools, including AlphaFold 2, the team built an integrative model of capsid assembly and designed structure-guided experiments. Their findings revealed that copia adopts a capsid fold similar to the mature HIV-1 capsid, reinforcing previous observations from purified transposable element structures.

Insights into the Retrotransposon Lifecycle

Klumpe and his colleagues also captured copia’s replication cycle within intact egg chambers. Like retroviruses, retrotransposons are transcribed from the genome, exported to the cytoplasm, and translated into proteins that form virus-like particles. These particles then reverse transcribe their RNA genome.

A major unanswered question in retrotransposon research is how these elements re-enter the nucleus to integrate new copies into the host genome. By examining copia capsids within cells, the researchers found that viral particles cluster near nuclear pores, which act as gateways between the cytoplasm and the nucleus.

Similar to its evolutionary relative HIV-1, copia likely enters the nucleus as an intact particle through these nuclear pores. The study suggests that the nuclear pore complex functions as a molecular sieve, permitting entry only to viral particles of a specific size. Genetic manipulations disrupting nuclear transport led to copia particles being retained in the cytoplasm, further supporting this hypothesis.

Transposon Regulation and Repression

While Drosophila harbors numerous retrotransposons, copia is among the most actively expressed. Previous research indicates that copia primarily targets the male germ line. In this study, researchers explored how Drosophila’s transposon repression system, the PIWI-piRNA pathway, silences copia.

In fruit fly testes, anti-sense piRNAs targeting copia were found in high abundance. In flies lacking an active piRNA pathway—where transposons are freely expressed—copia appeared in a non-viable state within female germ line nuclei destined for programmed cell death. However, in male flies, copia was observed moving from the cytoplasm to the gamete nucleus during spermatogenesis, suggesting that nuclear entry is a crucial step in its replication cycle within the male reproductive system.

Once dismissed as junk DNA, transposons are now recognized as having a profound impact on host biology and evolution. “Our study highlights the potential of cryo-ET to unravel the structural biology of transposons and provide new insights into the mechanisms governing their replication cycles,” says Sven Klumpe of the Max Planck Institute of Biochemistry.

Julius Brennecke, Senior Group Leader at IMBA, anticipates new research opportunities with Klumpe joining IMBA and the IMP. “Cryo-Electron Tomography is widely applicable to many biological questions, and Sven will find plenty of opportunities for collaboration across the Vienna BioCenter.”