Abstract
Mobile group II introns retrohome by an RNP-based mechanism in which the intron RNA reverse splices into a DNA site and is reverse transcribed by the associated intron-encoded protein. The resulting intron cDNA is then integrated into the genome by cellular mechanisms that have remained unclear. Here, we used an Escherichia coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify host factors that function in retrohoming. We then analyzed mutants identified in these and previous genetic screens by using a new biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro. The genetic and biochemical analyses indicate a retrohoming pathway involving degradation of the intron RNA template by a host RNase H and second-strand DNA synthesis by the host replicative DNA polymerase. Our results reveal ATP-dependent steps in both cDNA and second-strand synthesis and a surprising role for replication restart proteins in initiating second-strand synthesis in the absence of DNA replication. We also find an unsuspected requirement for host factors in initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming. Key features of the retrohoming mechanism may be used by human LINEs and other non-LTR-retrotransposons, which are related evolutionarily to mobile group II introns. Our findings highlight a new role for replication restart proteins, which function not only to repair DNA damage caused by mobile element insertion, but have also been co-opted to become an integral part of the group II intron retrohoming mechanism.
Highlights
Mobile group II introns are non-long-terminal-repeat retroelements that are commonly found in prokaryotes and in organellar genomes of eukaryotes and are thought to be evolutionary ancestors of splicesomal introns and retrotransposons in higher organisms [1]
They spread within and between genomes by a mechanism termed ‘‘retrohoming’’ in which the intron RNA inserts directly into a DNA site and is reverse transcribed by an intron-encoded reverse transcriptase
The resulting intron cDNA is integrated into the genome by host factors, but how it occurs has remained unclear
Summary
Mobile group II introns are non-long-terminal-repeat (nonLTR) retroelements that are commonly found in prokaryotes and in organellar genomes of eukaryotes and are thought to be evolutionary ancestors of splicesomal introns and retrotransposons in higher organisms [1]. These two components function together in a ribonucleoprotein complex (RNP) to promote intron mobility by a mechanism in which the excised intron lariat RNA uses its ribozyme activity to reverse splice directly into a DNA site and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the genome by host enzymes [2,3,4,5] By using this mechanism, group II introns insert at high frequency into specific DNA target sites in a process called ‘‘retrohoming’’ and at low frequency into ectopic sites that resemble the normal homing site in a process called ‘‘retrotransposition’’ or ‘‘ectopic retrohoming’’ [6]. Host factors that function in late steps in the retrohoming of group II intron lariat RNAs, the major retrohoming pathway used in nature, have not been identified conclusively in any organism, and the mechanisms used for these steps have remained poorly understood
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