Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, Precise RNA-mediated INsertion of Transgenes (PRINT), relies on co-delivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Conserved and divergent DNA recognition specificities and functions of R2 retrotransposon N-terminal domains.

R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon.

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

Evolution and dosage compensation of nucleolar organizing regions (NORs) mediated by mobile elements in turtles with female (ZZ/ZW) but not with male (XX/XY) heterogamety.

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.