Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation.
Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3' end of the intron RNA to the 5' end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5' junctions with 5' exon resections, 5' intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.
Zhuang
Fanglei
F
Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas, Austin, TX 78712, USA.
Mastroianni
Marta
M
White
Travis B
TB
Lambowitz
Alan M
AM
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D007438:Introns; D009865:Oocytes; D012313:RNA / Q000235:genetics*; D018626:Retroelements; D014982:Xenopus laevis / Q000235:genetics*
D018076:DNA, Complementary; D018626:Retroelements; D012313:RNA