Structures of human LINE-1 provide molecular insights into ‘copy’ and ‘paste’ retrotransposition process during target-primed reverse transcription
Around 17% of the human genome is made up of Long Interspersed Element 1 (LINE-1) sequences. This is a mobile element and, through the process called retrotransposition, it utilises an RNA intermediate to ‘copy’ and ‘paste’ itself from one location in our genomes to another. Approximately 100 LINE-1s retain the ability to retrotranspose, which is known to disrupt genes and cause DNA breakages, and is notably reactivated in many cancers. George Ghanim, a postdoc in Kelly Nguyen’s group in the LMB’s Structural Studies Division and now an Assistant Professor at Princeton University, US, has led a study to better understand how LINE-1 retrotransposes throughout the genome.
Propagation of LINE-1 occurs by a mechanism named target-primed reverse transcription (TPRT) and utilises the ORF2 protein (ORF2p) encoded by LINE-1. After entry into the nucleus, ORF2p severs one DNA strand at the target sequence and reverse transcribes LINE-1 messenger RNA into a new genomic location. This leaves a copy of LINE-1’s sequence on one DNA strand but, to fully complete insertion, LINE-1 must also follow this process on the second strand of DNA.
To understand the molecular mechanism behind target DNA restructuring for TPRT, George, assisted by Hongmiao Hu also in Kelly’s group, used electron cryo-microscopy (cryo-EM) to examine the structure of the ORF2p TPRT complex. The complex examined was biochemically stalled after making the first DNA cut and mid-initiation of RNA copying. The results were backed up by light microscopy experiments conducted by Kelly with analysis support from Jerome Boulanger, Senior Investigator Scientist in the LMB’s Light Microscopy Facility.
The team ultimately solved four structures of the complex, revealing that ORF2p extensively bends and unwinds the target DNA, effectively breaking the DNA in half. They also established that ORF2p does indeed also cut the second strand of DNA, nicking it during the reverse transcription stage of the first strand. This adds to the likeliness that ORF2p also subsequently completes the reverse transcription of the second strand.
It has previously been established that LINE-1 hijacks host cell machinery to facilitate its insertion into the genome. To better understand this, the team used AlphaFold3 structure prediction to identify potential binding sites for two cellular factors, proliferating cell nuclear antigen (PCNA) and poly(A)-binding protein 1 (PABPC1). The predicted binding site for PCNA and PABPC1 aligned with the target DNA position and RNA, respectively, in the team’s cryo-EM structures, suggesting that they play a mechanistically important role to regulate retrotransposition.
This research advances our understanding of LINE-1 retrotransposition, providing new insights into the extensive DNA remodelling by ORF2p during TPRT and affirming the mechanism behind the second strand cleavage. It also identifies novel binding sites for PCNA and PABPC1 on ORF2p, highlighting their potential role in LINE-1 retrotransposition. Furthering our understanding of LINE-1 retrotransposition is clinically important as there are 124 identified instances of germline LINE-1 retrotransposition events that have caused genetic diseases. Additionally, it is understood that somatic LINE-1 insertions frequently occur in many human cancers and can contribute to ageing, neurodegeneration, inflammation and the pathology of cancers more generally.
This work was funded by UKRI MRC, the Jane Coffin Childs Memorial Fund for Medical Research and EMBO.
George has recently launched his own independent research group at Princeton University where he will continue to investigate the structural mechanisms of retrotransposition.
Further references
Structural mechanism of LINE-1 target-primed reverse transcription. Ghanim, G.E., Hu, H., Boulanger, J., Nguyen, T.H.D. Science
Kelly’s group page
George Ghanim, Princeton University
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