Abstract
Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications.
Highlights
Mobile group II introns are site-specific retrotransposons that consist of a catalytically active intron RNA and an intron-encoded protein (IEP), with reverse transcriptase (RT) activity [1]
The finding of strong differential inhibition of linear relative to lariat intron retrohoming in D. melanogaster mutants indicates that the non-homologous end joining (NHEJ) factor ligase 4 (Lig4) is the predominant enzyme involved in ligating the intron cDNA to the upstream exon and that extra nucleotide addition by the DNA repair polymerase h (PolQ) plays a crucial role
Lig4 and PolQ appear to be the major enzymes playing these roles in D. melanogaster, residual linear intron RNA retrohoming with extra nucleotide addition occurs in both the lig4 and polQ mutants, indicating that other DNA ligases and polymerases can serve as backups that perform the same functions at lower efficiency
Summary
Mobile group II introns are site-specific retrotransposons that consist of a catalytically active intron RNA (ribozyme) and an intron-encoded protein (IEP), with reverse transcriptase (RT) activity [1]. Intron integration is targeted to the ligated-exon junction in an intronless alleles in a process called ‘‘retrohoming’’, but can occur at lower frequency into ectopic sites that resemble the homing site in a process called ‘‘retrotransposition’’ or ‘‘ectopic retrohoming’’. In both cases, the intron inserts into the new DNA site by a novel mechanism in which the excised intron lariat RNA fully reverse splices into a DNA strand and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the genome by host enzymes [1,10,11,12,13,14]. Retrohoming leads to the expansion of intron-containing alleles in a population, while ectopic retrohoming provides a means of intron dispersal to new sites
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
