Illuminating the transposon insertion landscape in plants using Cas9-targeted Nanopore sequencing and a novel pipeline

Transposable elements (TEs), which occupy significant portions of most plant genomes, are a major source of genomic novelty, contributing to plant adaptation, speciation and new cultivar production. The often large, complex genomes of plants make identifying TE insertions from short reads challenging, while whole-genome sequencing remains expensive. To expand the toolbox for TE identification in plants, we used the recently developed Cas9-targeted Nanopore sequencing (CANS) approach. Additionally, as no current bioinformatics tools automatically detect TE insertions after CANS, we developed NanoCasTE, a novel pipeline for target TE insertion discovery. We performed CANS of three copies of EVD retrotransposons in wild-type Arabidopsis thaliana and obtained up to 40x coverage of the targets after only a few hours of sequencing on a MinION sequencer. To estimate the ability to detect new TE insertions, we exploited the A. thaliana ddm1 mutant, which has elevated TE activity. Using CANS, we detected 84% of these insertions in ddm1 after generating only 4420 Nanopore reads (0.2x genome coverage), and also unambiguously identified their locations, demonstrating the method's sensitivity. CANS of pooled (~50 plants) ddm1 plants captured >800 EVD insertions, especially in centromeric regions. CANS also identified insertions of a Ty3/Gypsy retrotransposon in the genomes of two Aegilops tauschii plants, a species with a large genome.

Structure of Drosophila melanogaster ARC1 reveals a repurposed molecule with characteristics of retroviral Gag

The tetrapod neuronal protein ARC and its Drosophila melanogaster homolog, dARC1, have important but differing roles in neuronal development. Both are thought to originate through exaptation of ancient Ty3/Gypsy retrotransposon Gag, with their novel function relying on an original capacity for self-assembly and encapsidation of nucleic acids. Here, we present the crystal structure of dARC1 CA and examine the relationship between dARC1, mammalian ARC, and the CA protein of circulating retroviruses. We show that while the overall architecture is highly related to that of orthoretroviral and spumaretroviral CA, there are substantial deviations in both amino- and carboxyl-terminal domains, potentially affecting recruitment of partner proteins and particle assembly. The degree of sequence and structural divergence suggests that Ty3/Gypsy Gag has been exapted on two separate occasions and that, although mammalian ARC and dARC1 share functional similarity, the structures have undergone different adaptations after appropriation into the tetrapod and insect genomes.

Structure of D. melanogaster ARC1 reveals a repurposed molecule with characteristics of retroviral Gag

ABSTRACTThe tetrapod neuronal protein ARC and its D. melanogaster homologue, dARC1, have important but differing roles in neuronal development. Both are thought to originate through exaptation of ancient Ty3/Gypsy retrotransposon Gag genes, with their novel function relying on an original capacity for self-assembly and encapsidation of nucleic acids. Here, we present the crystal structure of dARC1 CA and examine the relationship between dARC1, mammalian ARC and the CA protein of circulating retroviruses. We show that whilst the overall architecture is highly related to that of orthoretroviral and spumaretroviral CA, there are significant deviations in both N- and C-terminal domains, potentially affecting recruitment of partner proteins and particle assembly. The degree of sequence and structural divergence suggests that Ty3/Gypsy Gag has been exapted on two separate occasions and that, although mammalian ARC and dARC1 share functional similarity, the structures have undergone different adaptations after appropriation into the tetrapod and insect genomes.

Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses

Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation. They encode a Gag polypeptide, the capsid domains of which can oligomerize to form a virus-like particle. The structures of retroviral capsids have been extensively described. They assemble an immature viral particle through oligomerization of full-length Gag. Proteolytic cleavage of Gag results in a mature, infectious particle. In contrast, the absence of structural data on LTR retrotransposon capsids hinders our understanding of their function and evolutionary relationships. Here, we report the capsid morphology and structure of the archetypal Gypsy retrotransposon Ty3. We performed electron tomography (ET) of immature and mature Ty3 particles within cells. We found that, in contrast to retroviruses, these do not change size or shape upon maturation. Cryo-ET and cryo-electron microscopy of purified, immature Ty3 particles revealed an irregular fullerene geometry previously described for mature retrovirus core particles and a tertiary and quaternary arrangement of the capsid (CA) C-terminal domain within the assembled capsid that is conserved with mature HIV-1. These findings provide a structural basis for studying retrotransposon capsids, including those domesticated in higher organisms. They suggest that assembly via a structurally distinct immature capsid is a later retroviral adaptation, while the structure of mature assembled capsids is conserved between LTR retrotransposons and retroviruses.

A Model System in S2 Cells to Test the Functional Activities of Drosophila Insulators

Insulators are a special class of regulatory elements that can regulate interactions between enhancers and promoters in the genome of high eukaryotes. To date, the mechanisms of insulator action remain unknown, which is primarily related to the lack of convenient model systems. We suggested studying a model system which is based on transient expression of a plasmid with an enhancer of the copia transposable element, in Drosophila embryonic cell lines. We demonstrated that during transient transfection of circle plasmids with a well-known Drosophila insulator from the gypsy retrotransposon, the insulator exhibits in an enhancer-blocking assay the same properties as in Drosophila stable transgenic lines. Therefore, the Drosophila cell line is suitable for studying the main activities of insulators, which provides additional opportunities for investigating the functional role of certain insulator proteins.