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The activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own terminal inverted repeat

ABSTRACT: The transposon piggyBat has already been used as a genomic tool for studies in human cells13. Here, through a combination of in vitro DNase I footprinting experiments with cell culture-based transposition assays, we have been able to rationalize the organization of the piggyBat TIRs. Based on this, we solved the cryo-EM structure of the piggyBat pre-synaptic complex at 3.4Å resolution. By combining these results, we discovered that piggyBat's transposition activity in vivo is severely restricted by a transposase binding site on its Left End (LE). Further modifications of the RE and the elimination of N-terminal phosphorylation sites of the transposase and the duplication of C-terminal site-specific DNA domain increased transposition activity by approximately two orders of magnitude relative to wild type. Taken together, the results indicate a previously unobserved down-regulation of transposon activity by a elongated TIR. The TIR modifications described here lead to a transposition activity increase comparable to the most highly active reported piggyBac version with no detectable change in chromosomal integration fidelity

ORGANISM(S): Homo sapiens

PROVIDER: GSE245531 | GEO | 2024/11/25

REPOSITORIES: GEO

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Project description:Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo ‘peel-and-paste’ transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB-family genes that are evolutionarily related to the CRISPR-associated effectors Cas9 and Cas12. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that IscB and TnpB are essential to prevent loss of the donor IS element and potential transposon extinction as a consequence of the TnpA transposition mechanism. We first performed phylogenetic analysis of IscB/TnpB proteins and selected a family of related IS elements from Geobacillus stearothermophilus that we predicted would be mobilized by a common TnpA homolog. After reconstituting transposition using a heterologous expression system in E. coli, we found that IS elements were readily lost from the donor site due to the activity of TnpA in rejoining the flanking sequences back together upon excision. However, these IS elements also encode non-coding RNAs that guide TnpB and IscB nucleases to precisely recognize and cleave these excision products, leading either to elimination of the excision product or re-installation of the transposon through recombination. Indeed, under experimental conditions in which TnpA and TnpB-RNA complexes were co-expressed together with a genomically integrated IS element, transposon retention was significantly increased relative to conditions expressing TnpA alone. Remarkably, both TnpA and TnpB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between transposase and nuclease. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.

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The activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own terminal inverted repeat

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